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Tumor suppressive ZBTB4 inhibits cell growth by regulating cell cycle progression and apoptosis in Ewing sarcoma
Biomedicine & Pharmacotherapy 100 (2018) 108–115
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Tumor suppressive ZBTB4 inhibits cell growth by regulating cell cycle progression and apoptosis in Ewing sarcoma
T
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Yongxin Yua, Ruguo Shanga, , Yunzhou Chena, Jiehua Lia, Zhichao Lianga, Jianwei Hua, Kai Liua, ⁎ Chao Chenb, a b
Guangzhou Orthopedics Hospital, 449 Dongfeng Road, Yuexiu District, Guangzhou, Guangdong Province, 510045, China Department of Orthopedics, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong Province 510515, China
A R T I C L E I N F O
A B S T R A C T
Keywords: ZBTB4 Survivin Ewing sarcoma Proliferation Apoptosis Cell cycle
Increasing studies identify that zinc finger and BTB domain containing 4 (ZBTB4) functions as a tumor suppressor in human cancer. Underexpression of ZBTB4 is correlated with poor survival of breast cancer patients. However, the expression of ZBTB4 and its possible function remain unknown in Ewing sarcoma (ES). To clarify these issues, we investigated the expression difference between ES and normal tissues based on Gene Expression Omnibus (GEO) data from R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). GEO data (GSE68776) indicated that the expression of ZBTB4 in ES tissues was prominently lower compare to normal tissues. Our data further confirmed the underexpression of ZBTB4 in ES tissues. GEO data (GSE63157 and GSE17679) demonstrated that ZBTB4 underexpression predicted a obvious shorter overall survival and eventfree survival of ES patients. Interestingly, the expression of ZBTB4 was inversely correlated with proliferation makers Ki-67 and proliferating cell nuclear antigen (PCNA) in ES tissues. In vitro, ZBTB4 overexpression inhibited cell proliferation, and induced cell cycle arrest at G1 phase and apoptosis in SK-ES-1 and RD-ES cells. Moreover, ZBTB4 restoration suppressed the tumor growth of ES in mice. An inversely correlation between ZBTB4 and Survivin expression was observed in ES tissues. ZBTB4 overexpression reduced Survivin abundance in ES cells. Notably, Survivin restoration reversed the regulatory effect of ZBTB4 on ES cell proliferation, cell cycle progression and apoptosis. To conclude, our data indicated that ZBTB4 exhibited a tumor suppressive role in ES possibly by reducing Survivin expression. ZBTB4/Survivin axis might serve as a therapeutic target for ES.
1. Introduction Ewing sarcoma (ES) is considered as the 2nd common pediatric malignant cancer in bone [1]. The osseous sites and extra skeletal soft tissues are main sources of ES [2,3]. Metastatic disease are presented in around 25–30% of ES patients at diagnosis [4]. As for early-stage ES patients, surgical resection, chemotherapy and radiotherapy are the primary therapeutic approaches [5,6]. After a period of treatment, longterm survival rate of early-stage ES patients is about 70% [5,6]. However, the survival rate is as low as 20–30% in patients with metastatic or recurrent disease, who are underwent systematic treatment [5,6]. Thus, it is critical to identify key molecular markers implicated in tumor metastasis and therapeutic resistance, and disclose novel therapeutic targets for patients with metastatic or recurrent ES. Zinc finger and BTB domain containing 4 (ZBTB4), a mammalian
DNA-binding protein, contains C2H2 zinc fingers and a BTB/POZ domain and functions as a transcriptional repressor [7,8]. Accumulating studies have reported the tumor suppressive role of ZBTB4 in human cancer. ZBTB4 underexpression has been observed in several types of cancer including breast cancer, prostate cancer and neuroblastoma [8–11]. ZBTB4 is responsible for p53 induced apoptosis and cell cycle arrest via regulation of cyclin dependent kinase inhibitor 1A (CDKN1A) [8]. miR-17-92 cluster regulation of ZBTB4 represses the expression of specificity protein (Sp) transcription factors and suppresses breast cancer cell growth and invasion in vitro [9]. Furthermore, ZBTB4 is involved in reactive oxygen species (ROS) induced repression of SP in bladder cancer cells [12]. Several recent studies report that ROS inducing anticancer agents exert treating effect via induction of the Sp transcriptional repressor ZBTB4 [13–17]. ZBTB4 functions as a tumor suppressor in chronic lymphocytic leukemia (CLL) cells [18]. Drug
Abbreviations: ES, Ewing sarcoma; ZBTB4, zinc finger and BTB domain containing 4; CDKN1A, cyclin dependent kinase inhibitor 1A; ROS, reactive oxygen species; CLL, chronic lymphocytic leukemia; GEO, gene expression omnibus; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gelelectrophoresis; CCK-8, cell counting kit-8; PI, propidium iodide; PCNA, proliferating cell nuclear antigen ⁎ Corresponding authors. E-mail addresses: [email protected] (R. Shang), [email protected] (C. Chen). https://doi.org/10.1016/j.biopha.2018.01.132 Received 5 December 2017; Received in revised form 21 January 2018; Accepted 28 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. The expression difference of ZBTB4 in ES and normal tissues. A) GEO data (GSE68776) from R2: Genomics Analysis and Visualization Platform (http://r2.amc. nl) indicated that the expression of ZBTB4 in ES tissues was significantly lower than that in normal tissues. B) qRT-PCR analysis of ZBTB4 expression between 14 ES tissues and 4 normal bone tissues. C) Four pairs of ES and normal bone tissues detected by immunoblotting for ZBTB4 expression. Quantitative data confirmed ZBTB4 underexpression in ES tissues.
Fig. 2. The prognostic significance of ZBTB4 in ES patients. Different subgroups (ZBTB4 low/high expression) were plotted according to the cutoff values of ZBTB4, which were defined as the median of the cohort. GEO data (GSE63157 and GSE17679) revealed that ES patients with low ZBTB4 level showed a significant shorter overall survival and event-free survival compared to cases with high ZBTB4 level.
2. Materials and methods
induced ZBTB4 upregulation by targeting miRNAs inhibits tumor growth of prostate cancer in vivo [19]. Recent study figures out that loss of ZBTB4 leads to genomic instability and induces skin carcinogenesis under DMBA/TPA treatment [7]. However, there is no systematic study about the expression and role of ZBTB4 in ES. This study carried out a systematic analysis about the expression and prognostic significance of ZBTB4 based on Gene Expression Omnibus (GEO) data. Then, we investigated the role of ZBTB4 and its underlying mechanism in ES cells. We found that ZBTB4 underexpression predicts poor prognosis of ES patients. ZBTB4 inhibited proliferation of ES cells via inducing apoptosis and cell cycle arrest. Survivin might mediate the tumor suppressive role of ZBTB4 in ES. Our data possibly provide experimental and theoretical basis for ES therapeutic improvement.
2.1. ES samples, cell lines and transfection A total of 14 ES specimens were collected from patients’ resected tumor at our hospital. Inclusion criteria as follows: patients with pathological diagnosis; without radiotherapy, chemotherapy or biological treatment before operation. Four specimen of normal bones were obtained from patients treated with femoral-head replacement. All samples were immediately frozen in liquid nitrogen and stored at −80 °C. Written informed consent was obtained before collection. The study was approved by Ethics Committee of Southern Medical University. The ES cell lines, SK-ES-1 and RD-ES, were purchased from ATCC (Rockville, Maryland, USA). Cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and antibiotics (100 109
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Fig. 3. ZBTB4 overexpression inhibits the proliferation of ES cells. A) GEO data (GSE34620) indicated that ZBTB4 expression was inversely correlated with proliferation markers Ki-67 and PCNA levels in ES tissues. n = 117. B) SK-ES-1 and RD-ES cells that were transduced with retro-ZBTB4 or empty vector (EV) were subjected to western blotting for ZBTB4 expression. n = 3, *P < .05. C) ZBTB4 overexpression prominently reduced the proliferation capacity of ES cells. n = 3, *P < .05.
units/ml penicillin and 100 μg/ml streptomycin; Sigma-Aldrich, St. Louis, MO, USA), maintained at 37 °C in 5% CO2. Retroviral vector pMMP-ZBTB4 was generated, packaged and transduced as described [20]. Survivin expression plasmid (pcDNA3.1-survivin) and empty vector were purchased from GeneCopoeia (Guangzhou, China). ZBTB4 siRNA (sc-93593) and scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Transfection was carried out by Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
GCGTACAG-3′.
2.3. Western blotting Protein of cell lines, cancer and adjacent tissues was extracted with RIPA lysis buffer and its concentration was measured in accordance with BCA kit (Pierce, Rockford, IL, USA). All the samples that were quantified as 5 μg to 15 μg protein each lane were transferred into PVDF membrane after separating by 4–15% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). After that, the membrane was sealed with 5% skimmed milk, and incubated overnight at 4 ℃ with primary antibodies including ZBTB4 (sc-514883; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Survivin (ab76424, Abcam, Cambridge, MA, USA), Ki-67 (sc-23900, Santa Cruz Biotechnology), PCNA (#13110, Cell Signaling Technology, Beverly, MA, USA), Sp1 (sc420, Santa Cruz Biotechnology), Sp3 (sc-136479, Santa Cruz Biotechnology), Sp4 (sc-390124, Santa Cruz Biotechnology) and GAPDH (sc‑47,724; Santa Cruz Biotechnology), and then incubated for 2 h at room temperature with secondary antibodies (#7076 and #7074; Cell Signaling Technology). A chemiluminescent detection system (BioRad, Hercules,CA, USA) was used for imaging.
2.2. RNA extraction and quantitative real-time PCR Total RNA of ES tissues were extracted with Trizol Reagent (Invitrogen). And cDNA was reversed with using Transcript FirststrandcDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). By using an iCycler RT-PCR system (Bio-Rad, Munich, Germany), quantitative real-time PCR could be completed by using SuperReal PreMix SYBR Green (FP204-02, TIANGEN, Beijing, China). The primers for ZBTB4 and actin were as follows: ZBTB4 forward 5′-AGGAAGTACCCC TGCCGCTA-3′, reverse 5′-TTGTAGCCTCCATTGGGTGT-3′; actin forward 5′-GGACTTCGAGCAAGAGATGG-3′, reverse 5′-AGCACTGTGTTG 110
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Fig. 4. ZBTB4 overexpression leads to apoptosis and cell cycle arrest of ES cells. A) ZBTB4 overexpression increased the portion of apoptotic ES cells. n = 3, *P < .05. B) ZBTB4 overexpression induced cell cycle arrest at G1 phase in both SK-ES-1 and RD-ES cells. n = 3, *P < .05.
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Fig. 5. ZBTB4 overexpression suppresses the tumor growth of ES in mice. SK-ES-1 cells that were transduced with retor-ZBTB4 or empty vector (EV) were subcutaneously implanted into nude mice. Tumor growth curves indicated that ZBTB4 overexpression restrained the tumor growth of ES in vivo. n = 5.
tissues based on GEO data from R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). GEO data (GSE68776) revealed that the expression ZBTB4 mRNA in ES tissues was significant lower than that in normal tissues (P = .0146, Fig. 1A). Then, investigation of our ES and normal bone tissues further confirmed underexpression of both ZBTB mRNA and protein in ES tissues compared to normal tissues (P < .0001 and P = .0017, respectively, Fig. 1B and C). The prognostic significance of ZBTB4 was disclosed using GEO data. GEO data (GSE63157 and GSE17679) suggested that ZBTB4 underexpression predicted a significant shorter overall survival and eventfree survival of ES patients (P < .05, respectively, Fig. 2). Taken together, we found that ZBTB4 was a promising prognostic biomarker for ES patients.
2.4. Cell counting kit-8 (CCK-8) assay The proliferation of ES cells was measured by CCK-8 assay. ES cells were seeded in a 96-well plates for cell culture after transfection. The specific steps as follows: each well was added with 10 μl CCK-8 reagent, fully mixed, placed in 37℃ incubator for 1 h. The optical density at 450nm of each well was measured with a Microplate reader (Bio-Rad). 2.5. Cell apoptosis assay The apoptosis of ES cells was determined by Annexin V/propidium iodide (PI) assay. After cell collection, the solution was prepared according to the instruction of Annexin V/PI method. The concentration of the cells was adjusted to 5 × 105/ml. Each tube was added with Annexin V-FITC and PI (Roche Molecular Biochemicals, Indianapolis, IN, USA), mixed and shielded from light for 10 min. Finally, the cells were added with 300 μl binding buffer and detected by flow cytometer (Beckman Coulter, Brea, CA, USA).
3.2. ZBTB4 restoration suppresses proliferation, and induces cell cycle arrest and apoptosis in ES cells
The ES cells (2 × 106 cells) that were transduced with retro-ZBTB4 or retro-EV were injected subcutaneously into the right flank of sixweek-old female nude mice. Tumor size were measured every 4 days after subcutaneous injection. Tumor volume (mm3) = longer diameter × (shorter diameter)2/2. All animal experiments were approved by the Ethics Committee of Southern Medical University.
Next, we were aimed to determine the regulatory effect of ZBTB4 on cell proliferation, cell cycle progression and apoptosis of ES cells. We first investigated the correlation between the expression of ZBTB4 and proliferation makers including Ki-67 and proliferating cell nuclear antigen (PCNA) using GEO data (GSE34620). Notably, ZBTB4 expression was inversely correlated with Ki-67 and PCNA levels in ES tissues (r = −0.480, P < .0001 and r = −0.469, P < .0001, Fig. 3A). Then, ZBTB4 was overexpressed in SK-ES-1 and RD-ES cells via retroviruses infection (P < .05, respectively, Fig. 3B). CCK-8 assay indicated that ZBTB4 restoration prominently inhibited proliferation of SK-ES-1 and RD-ES cells in vitro (P < .05, respectively, Fig. 3C). Furthermore, ZBTB4 overexpression obviously induced apoptosis of SK-ES-1 and RDES cells (P < .05, respectively, Fig. 4A). ZBTB4 restoration led to cell cycle arrest at G1 phase in both ES cell lines (P < .05, respectively, Fig. 4B). ZBTB4 overexpression increased chemosensitivity to doxorubicin, part of the regular chemotherapeutic regimen for Ewing sarcoma patients, in both SK-ES-1 and RD-ES cells. (P < .05, respectively, Supplementary Fig. 1). Thus, our data suggested that ZBTB4 prohibited cell proliferation via inducing cell cycle arrest and apoptosis in ES.
2.8. Statistical analysis
3.3. ZBTB4 restoration inhibits tumor growth of ES in mice
Measurement data were expressed as mean ± standard deviation. Statistical analysis was done by GraphPad Prism 5 software (GraphPad Software, Inc, San Diego, CA, USA) using t-test, ANOVA, Kaplan-Meier method, Log-rank test and Spearman’s rank correlation analysis, respectively. P < .05 was considered statistically significant. 3. Results
Based on our previous findings, we further invested whether ZBTB4 restoration inhibited tumor growth of ES in vivo. ZBTB4 overexpressing SK-ES-1 cells and control cells were subcutaneously implanted into nude mice, respectively. Tumor growth curves indicated that the tumor size in ZBTB overexpression group was significantly smaller than that in control group (P < .05, Fig. 5). Altogether, we found that ZBTB4 overexpression restrained the growth of ES cells in vitro and in vivo.
3.1. The expression of ZBTB4 and its prognostic significance in ES tissues
3.4. Survivin mediates the tumor suppressive role of ZBTB4 in ES cells
2.6. Cell cycle analysis Transfected ES cells were harvested and centrifuged with 1000 g for 5 min. Cells were washed with PBS and fixed with ethanol for 1 h at 4 °C. After washing with PBS for three times, cells were resuspended in 0.2 ml of RNase A buffer (1 mg/ml) at 37 °C for 30 min. Then cells were stained with 0.3 ml PI buffer (50μl/ml). The cell cycle distribution of ES cells by using the flow cytometer (FACSCalibur). 2.7. Tumor formation assay
Firstly, we investigated the expression of ES in ES and normal
Since previous studies report that ZBTB4 decreases Sp genes and 112
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Fig. 6. Survivin mediates the tumor suppressive role of ZBTB in ES cells. A) GEO data (GSE34620) indicated an inverse correlation between ZBTB4 and Survivin expression in ES tissues. n = 117. B) ZBTB4 overexpression obviously reduced Survivin abundance in both SK-ES-1 and RD-ES cells. n = 3, *P < .05. C) SK-ES-1 cells that were tranfected with indicated vectors were subjected to immunoblotting for Survivin expression. n = 3, *P < .05 vs EV group, #P < .05 vs ZBTB4 group. D) Survivin restoration enhanced the proliferation of ZBTB4 overexpressing SK-ES-1 cells. n = 3, *P < .05 vs EV group, #P < 0.05 vs ZBTB4 group. E) Survivin restoration reversed the effect of ZBTB4 overexpression on the apoptosis of SK-ES-1 cells. n = 3, *P < .05 vs EV group, #P < .05 vs ZBTB4 group. F) Survival restoration abolished the inhibitory effect of ZBTB4 overexpression on cell cycle progression in SK-ES-1 cells. n = 3, *P < .05 vs EV group, #P < .05 vs ZBTB4 group.
(Supplementary Fig. 3). While, ZBTB4 knockdown increased the expression of Survivin, Ki-67 and PCNA in RD-ES cells (Supplementary Fig. 4). Survivin is a critical regulator of cell growth and apoptosis in ES [21–23]. To clarify whether ZBTB4 plays a tumor suppressive role by targeting Survivin, Survivin expression was restored in ZBTB4 overexpressing SK-ES-1 cells via plasmid transfection (P < .05, Fig. 6C). Interestingly, Survivin restoration abolished the regulatory effect of ZBTB4 on proliferation, cell cycle progression and apoptosis in SK-ES-1 cells (P < .05, respectively, Fig. 6D–F). Taken together, ZBTB4 regulated proliferation, cell cycle progression and apoptosis at least partially
subsequent reduces Sp-related oncogenes including Survivin [9,12]. Thus, we investigated the correlation between ZBTB4 and Survivin expression in ES tissues. GEO data (GSE34620) indicated an inverse correlation between ZBTB4 and Survivin expression in ES tissues (r = −0.521, P < .0001, Fig. 6A). Then, western blotting analysis revealed that ZBTB4 overexpression led to decrease of Sp1, Sp3 and Sp4 in SK-ES-1 and RD-ES cells (Supplementary Fig. 2). Furthermore, ZBTB4 restoration reduced Survivin level in both SK-ES-1 and RD-ES cells (P < .05, respectively, Fig. 6B). And the expression of Survivin in ZBTB4 restored xenograft tissues is obviously lower than control group 113
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cycle arrest at G1 phase and apoptosis of ES cells. ZBTB4 overexpression inhibited tumor growth in vivo. Further studies show that tumor suppressive ZBTB4 reduced Survivin abundance in ES cells. Survivin at least partially mediated the tumor suppressive role of ZBTB4 in ES cells. Our findings suggested that ZBTB4 might contribute to the development of targeted therapy of ES.
through targeting Survivin in ES cells. 4. Discussion We have identified that ZBTB4 was under-expressed in ES tissues compared to normal tissues. ZBTB4 underexpression was a significant indicator for poor prognosis of ES patients. Restoration of ZBTB4 inhibited the proliferation, and induced cell cycle arrest at G1 phase and apoptosis of ES cells in vitro possibly by reducing Survivin expression. In addition, the overexpression of ZBTB4 prohibited tumor growth in vivo. Loss or underexpression of tumor suppressors is closely associated with human cancer development and progression. The aberrant expression of ZBTB4 has been shown to facilitate the progression of breast cancer [9], prostate cancer [19] and neuroblastoma [11]. Yet, the under-expression of ZBTB4 has also been spotted in skin carcinogenesis recently [7]. We found that ZBTB4 was down-regulated in ES tissues, which was consistent with GEO data involved in ES. ZBTB4 has been recognized as a novel prognostic marker for breast cancer patients [9]. Our study also confirmed that ZBTB4 underexpression contributes to shorter survival of ES patients based on two different GEO data. To date, several studies show that ZBTB4 regulates several cellular processes in human cancer. For instance, ZBTB4 restoration suppresses the capacity of breast cancer cell growth and invasion [9]. ZBTB4 is responsible for p53 induced apoptosis and cell cycle arrest via regulation of CDKN1A [8]. Drug induced ZBTB4 upregulation by targeting miRNAs inhibits tumor growth of prostate cancer in vivo [19]. Our study found that ZBTB4 expression was inversely correlated with proliferation markers, Ki-67 and PCNA. ZBTB4 knockdown increased the levels of Ki-67 and PCNA in ES cells. ZBTB4 overexpression inhibited proliferation, and led to cell cycle arrest at G1 phase and increased apoptosis of ES cells. Moreover, ZBTB4 restoration obviously slowed down the tumor growth in mice. Thus, we suggest that ZBTB4 functions as a tumor suppressor via regulating proliferation, cell cycle progression and apoptosis in ES. Recent studies report that ROS inducing anticancer agents exert treating effect via induction of the Sp transcriptional repressor ZBTB4 [13–17]. Our data showed that ZBTB4 ZBTB4 overexpression increased sensitivity to doxorubicin in ES cells. Thus, ZBTB4 may be a potential therapeutic target for ES. Survivin plays an important role in cancer initiation, development and metastasis [24,25]. Several studies have reported that Survivin overexpression is correlated with tumor grade and prognosis [26,27]. Survivin has been found to be up-regulated in a plenty of cancers including hepatocellular carcinoma, breast cancer, gastric cancer etc [28–30]. Previous studies report that ZBTB4 decreases Sp genes and subsequent reduces Sp-related oncogenes including Survivin [9,12]. Consistently, in this study, ZBTB4 was inversely correlated with Survivin expression in ES tissues. ZBTB4 overexpression reduced Sp1, Sp3, Sp4 and Survivin abundance in ES cells. And ZBTB4 knockdown increased Survivin expression in RD-ES cells. Plus, we confirmed that Survivin was a downstream mediator of ZBTB4. Taken together, ZBTB4/Survivin axis works closely during ES tumorigenesis. ZBTB4 restrains ES progression possibly by inhibiting Survivin expression. In summary, we reveal a novel function of ZBTB4 in suppressing proliferation and inducing cell cycle arrest and apoptosis of ES cells by targeting Survivin. Nevertheless, there are some limitations in this study. The underlying molecular mechanism of the down-regulation of ZBTB4 and the target relationship between ZBTB4 and Survivin in ES cells still need further investigation. Moreover, ZBTB4, as a novel biomarker and target for treatment, remains to be further explored at clinical level.
Conflicts of interest All authors declare no conflicts of interest. Acknowledgement This study was supported by the Major Projects for Industry-university-research Collaborative Innovation of Science and Technology Plan of Guangzhou (no. 201604020095), Natural Science Foundation of Guangdong Province (no. 2015A030313367), Project of Administration of Traditional Chinese Medicine of Guangdong Province (no.20161164) and 13th Five-Year the Province Project by the Guangdong Provincial Administration of Traditional Chinese Medicine Management. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2018.01.132. References [1] N. Riggi, I. Stamenkovic, The biology of Ewing sarcoma, Cancer Lett. 254 (1) (2007) 1–10. [2] E.E. Karski, K.K. Matthay, J.M. Neuhaus, R.E. Goldsby, S.G. Dubois, Characteristics and outcomes of patients with Ewing sarcoma over 40 years of age at diagnosis, Cancer Epidemiol. 37 (1) (2013) 29–33. [3] Y.S. Lau, I.E. Adamopoulos, A. Sabokbar, H. Giele, C.L. Gibbons, N.A. Athanasou, Cellular and humoral mechanisms of osteoclast formation in Ewing’s sarcoma, Br. J. Cancer 96 (11) (2007) 1716–1722. [4] N. Esiashvili, M. Goodman, R.B. Marcus Jr., Changes in incidence and survival of Ewing sarcoma patients over the past 3 decades: surveillance epidemiology and end results data, J. Pediatr. Hematol. Oncol. 30 (6) (2008) 425–430. [5] N. Gaspar, D.S. Hawkins, U. Dirksen, I.J. Lewis, S. Ferrari, M.C. Le Deley, H. Kovar, R. Grimer, J. Whelan, L. Claude, O. Delattre, M. Paulussen, P. Picci, K. Sundby Hall, H. van den Berg, R. Ladenstein, J. Michon, L. Hjorth, I. Judson, R. Luksch, M.L. Bernstein, P. Marec-Berard, B. Brennan, A.W. Craft, R.B. Womer, H. Juergens, O. Oberlin, Ewing sarcoma: current management and future approaches through collaboration, J. Clin. Oncol. 33 (27) (2015) 3036–3046. [6] Y. Jiang, J. Ludwig, F. Janku, Targeted therapies for advanced Ewing sarcoma family of tumors, Cancer Treat. Rev. 41 (5) (2015) 391–400. [7] A. Roussel-Gervais, I. Naciri, O. Kirsh, L. Kasprzyk, G. Velasco, G. Grillo, P. Dubus, P.A. Defossez, Loss of the methyl-CpG-binding protein ZBTB4 alters mitotic checkpoint, increases aneuploidy, and promotes tumorigenesis, Cancer Res. 77 (1) (2017) 62–73. [8] A. Weber, J. Marquardt, D. Elzi, N. Forster, S. Starke, A. Glaum, D. Yamada, P.A. Defossez, J. Delrow, R.N. Eisenman, H. Christiansen, M. Eilers, Zbtb4 represses transcription of P21CIP1 and controls the cellular response to p53 activation, EMBO J. 27 (11) (2008) 1563–1574. [9] K. Kim, G. Chadalapaka, S.O. Lee, D. Yamada, X. Sastre-Garau, P.A. Defossez, Y.Y. Park, J.S. Lee, S. Safe, Identification of oncogenic microRNA-17-92/ZBTB4/ specificity protein axis in breast cancer, Oncogene 31 (8) (2012) 1034–1044. [10] H. Ross-Adams, A.D. Lamb, M.J. Dunning, S. Halim, J. Lindberg, C.M. Massie, L.A. Egevad, R. Russell, A. Ramos-Montoya, S.L. Vowler, N.L. Sharma, J. Kay, H. Whitaker, J. Clark, R. Hurst, V.J. Gnanapragasam, N.C. Shah, A.Y. Warren, C.S. Cooper, A.G. Lynch, R. Stark, I.G. Mills, H. Gronberg, D.E. Neal, P.S.G. CamCa, Integration of copy number and transcriptomics provides risk stratification in prostate cancer: a discovery and validation cohort study, EBioMedicine 2 (9) (2015) 1133–1144. [11] D. Yamada, R. Perez-Torrado, G. Filion, M. Caly, B. Jammart, V. Devignot, N. Sasai, P. Ravassard, J. Mallet, X. Sastre-Garau, M.L. Schmitz, P.A. Defossez, The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4, Oncogene 28 (27) (2009) 2535–2544. [12] G. Chadalapaka, I. Jutooru, S. Safe, Celastrol decreases specificity proteins (Sp) and fibroblast growth factor receptor-3 (FGFR3) in bladder cancer cells, Carcinogenesis 33 (4) (2012) 886–894. [13] K. Karki, E. Hedrick, R. Kasiappan, U.H. Jin, S. Safe, Piperlongumine induces reactive oxygen species (ROS)-dependent downregulation of specificity protein transcription factors, Cancer Prev. Res. (Phila.) 10 (8) (2017) 467–477. [14] E. Hedrick, X. Li, S. Safe, Penfluridol represses integrin expression in breast cancer through induction of reactive oxygen species and downregulation of Sp
5. Conclusion In summary, this study revealed that ZBTB4 was significantly downregulated in ES tissues, such correlated with shorter survival of ES patients. Functionally, ZBTB4 suppressed proliferation, and induced cell 114
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transcription factors, Mol. Cancer Ther. 16 (1) (2017) 205–216. [15] I. Jutooru, A.S. Guthrie, G. Chadalapaka, S. Pathi, K. Kim, R. Burghardt, U.H. Jin, S. Safe, Mechanism of action of phenethylisothiocyanate and other reactive oxygen species-inducing anticancer agents, Mol. Cell. Biol. 34 (13) (2014) 2382–2395. [16] E. Hedrick, L. Crose, C.M. Linardic, S. Safe, Histone deacetylase inhibitors inhibit rhabdomyosarcoma by reactive oxygen species-dependent targeting of specificity protein transcription factors, Mol. Cancer Ther. 14 (9) (2015) 2143–2153. [17] R. Kasiappan, I. Jutooru, K. Karki, E. Hedrick, S. Safe, Benzyl isothiocyanate (BITC) induces reactive oxygen species-dependent repression of STAT3 protein by downregulation of specificity proteins in pancreatic cancer, J. Biol. Chem. 291 (53) (2016) 27122–27133. [18] R. Bomben, S. Gobessi, M. Dal Bo, S. Volinia, D. Marconi, E. Tissino, D. Benedetti, A. Zucchetto, D. Rossi, G. Gaidano, G. Del Poeta, L. Laurenti, D.G. Efremov, V. Gattei, The miR-17 approximately 92 family regulates the response to toll-like receptor 9 triggering of CLL cells with unmutated IGHV genes, Leukemia 26 (7) (2012) 1584–1593. [19] K. Kim, G. Chadalapaka, S.S. Pathi, U.H. Jin, J.S. Lee, Y.Y. Park, S.G. Cho, S. Chintharlapalli, S. Safe, Induction of the transcriptional repressor ZBTB4 in prostate cancer cells by drug-induced targeting of microRNA-17-92/106b-25 clusters, Mol. Cancer Ther. 11 (9) (2012) 1852–1862. [20] K. Tu, J. Li, V.K. Verma, C. Liu, D.D. Billadeau, G. Lamprecht, X. Xiang, L. Guo, R. Dhanasekaran, L.R. Roberts, V.H. Shah, N. Kang, Vasodilator-stimulated phosphoprotein promotes activation of hepatic stellate cells by regulating Rab11-dependent plasma membrane targeting of transforming growth factor beta receptors, Hepatology 61 (1) (2015) 361–374. [21] S. Shelake, U.T. Sankpal, W. Paul Bowman, M. Wise, A. Ray, R. Basha, Targeting
[22]
[23]
[24] [25] [26] [27]
[28]
[29]
[30]
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specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting ewing sarcoma cell growth, Invest. New Drugs 35 (2) (2017) 158–165. B. Greve, F. Sheikh-Mounessi, B. Kemper, I. Ernst, M. Gotte, H.T. Eich, Survivin, a target to modulate the radiosensitivity of Ewing’s sarcoma, Strahlenther. Onkol. 188 (11) (2012) 1038–1047. O.M. Tirado, S. Mateo-Lozano, V. Notario, Roscovitine is an effective inducer of apoptosis of Ewing’s sarcoma family tumor cells in vitro and in vivo, Cancer Res. 65 (20) (2005) 9320–9327. X. Chen, N. Duan, C. Zhang, W. Zhang, Survivin and tumorigenesis: molecular mechanisms and therapeutic strategies, J. Cancer 7 (3) (2016) 314–323. M. Mobahat, A. Narendran, K. Riabowol, Survivin as a preferential target for cancer therapy, Int. J. Mol. Sci. 15 (2) (2014) 2494–2516. P.K. Jaiswal, A. Goel, R.D. Mittal, Survivin: a molecular biomarker in cancer, Indian J. Med. Res. 141 (4) (2015) 389–397. W. Feng, D. Cai, B. Zhang, G. Lou, X. Zou, Combination of HDAC inhibitor TSA and silibinin induces cell cycle arrest and apoptosis by targeting survivin and cyclinB1/ Cdk1 in pancreatic cancer cells, Biomed. Pharmacother. 74 (2015) 257–264. B. Tang, X. Liang, F. Tang, J. Zhang, S. Zeng, S. Jin, L. Zhou, Y. Kudo, G. Qi, Expression of USP22 and survivin is an indicator of malignant behavior in hepatocellular carcinoma, Int. J. Oncol. 47 (6) (2015) 2208–2216. S. Li, L. Wang, Y. Meng, Y. Chang, J. Xu, Q. Zhang, Increased levels of LAPTM4B, VEGF and survivin are correlated with tumor progression and poor prognosis in breast cancer patients, Oncotarget 8 (25) (2017) 41282–41293. Y. Gu, S. Jin, F. Wang, Y. Hua, L. Yang, Y. Shu, Z. Zhang, R. Guo, Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer, Biomed. Pharmacother. 68 (4) (2014) 471–475.
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Tumor suppressive ZBTB4 inhibits cell growth by regulating cell cycle progression and apoptosis in Ewing sarcoma
T
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Yongxin Yua, Ruguo Shanga, , Yunzhou Chena, Jiehua Lia, Zhichao Lianga, Jianwei Hua, Kai Liua, ⁎ Chao Chenb, a b
Guangzhou Orthopedics Hospital, 449 Dongfeng Road, Yuexiu District, Guangzhou, Guangdong Province, 510045, China Department of Orthopedics, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou, Guangdong Province 510515, China
A R T I C L E I N F O
A B S T R A C T
Keywords: ZBTB4 Survivin Ewing sarcoma Proliferation Apoptosis Cell cycle
Increasing studies identify that zinc finger and BTB domain containing 4 (ZBTB4) functions as a tumor suppressor in human cancer. Underexpression of ZBTB4 is correlated with poor survival of breast cancer patients. However, the expression of ZBTB4 and its possible function remain unknown in Ewing sarcoma (ES). To clarify these issues, we investigated the expression difference between ES and normal tissues based on Gene Expression Omnibus (GEO) data from R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). GEO data (GSE68776) indicated that the expression of ZBTB4 in ES tissues was prominently lower compare to normal tissues. Our data further confirmed the underexpression of ZBTB4 in ES tissues. GEO data (GSE63157 and GSE17679) demonstrated that ZBTB4 underexpression predicted a obvious shorter overall survival and eventfree survival of ES patients. Interestingly, the expression of ZBTB4 was inversely correlated with proliferation makers Ki-67 and proliferating cell nuclear antigen (PCNA) in ES tissues. In vitro, ZBTB4 overexpression inhibited cell proliferation, and induced cell cycle arrest at G1 phase and apoptosis in SK-ES-1 and RD-ES cells. Moreover, ZBTB4 restoration suppressed the tumor growth of ES in mice. An inversely correlation between ZBTB4 and Survivin expression was observed in ES tissues. ZBTB4 overexpression reduced Survivin abundance in ES cells. Notably, Survivin restoration reversed the regulatory effect of ZBTB4 on ES cell proliferation, cell cycle progression and apoptosis. To conclude, our data indicated that ZBTB4 exhibited a tumor suppressive role in ES possibly by reducing Survivin expression. ZBTB4/Survivin axis might serve as a therapeutic target for ES.
1. Introduction Ewing sarcoma (ES) is considered as the 2nd common pediatric malignant cancer in bone [1]. The osseous sites and extra skeletal soft tissues are main sources of ES [2,3]. Metastatic disease are presented in around 25–30% of ES patients at diagnosis [4]. As for early-stage ES patients, surgical resection, chemotherapy and radiotherapy are the primary therapeutic approaches [5,6]. After a period of treatment, longterm survival rate of early-stage ES patients is about 70% [5,6]. However, the survival rate is as low as 20–30% in patients with metastatic or recurrent disease, who are underwent systematic treatment [5,6]. Thus, it is critical to identify key molecular markers implicated in tumor metastasis and therapeutic resistance, and disclose novel therapeutic targets for patients with metastatic or recurrent ES. Zinc finger and BTB domain containing 4 (ZBTB4), a mammalian
DNA-binding protein, contains C2H2 zinc fingers and a BTB/POZ domain and functions as a transcriptional repressor [7,8]. Accumulating studies have reported the tumor suppressive role of ZBTB4 in human cancer. ZBTB4 underexpression has been observed in several types of cancer including breast cancer, prostate cancer and neuroblastoma [8–11]. ZBTB4 is responsible for p53 induced apoptosis and cell cycle arrest via regulation of cyclin dependent kinase inhibitor 1A (CDKN1A) [8]. miR-17-92 cluster regulation of ZBTB4 represses the expression of specificity protein (Sp) transcription factors and suppresses breast cancer cell growth and invasion in vitro [9]. Furthermore, ZBTB4 is involved in reactive oxygen species (ROS) induced repression of SP in bladder cancer cells [12]. Several recent studies report that ROS inducing anticancer agents exert treating effect via induction of the Sp transcriptional repressor ZBTB4 [13–17]. ZBTB4 functions as a tumor suppressor in chronic lymphocytic leukemia (CLL) cells [18]. Drug
Abbreviations: ES, Ewing sarcoma; ZBTB4, zinc finger and BTB domain containing 4; CDKN1A, cyclin dependent kinase inhibitor 1A; ROS, reactive oxygen species; CLL, chronic lymphocytic leukemia; GEO, gene expression omnibus; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gelelectrophoresis; CCK-8, cell counting kit-8; PI, propidium iodide; PCNA, proliferating cell nuclear antigen ⁎ Corresponding authors. E-mail addresses: [email protected] (R. Shang), [email protected] (C. Chen). https://doi.org/10.1016/j.biopha.2018.01.132 Received 5 December 2017; Received in revised form 21 January 2018; Accepted 28 January 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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Fig. 1. The expression difference of ZBTB4 in ES and normal tissues. A) GEO data (GSE68776) from R2: Genomics Analysis and Visualization Platform (http://r2.amc. nl) indicated that the expression of ZBTB4 in ES tissues was significantly lower than that in normal tissues. B) qRT-PCR analysis of ZBTB4 expression between 14 ES tissues and 4 normal bone tissues. C) Four pairs of ES and normal bone tissues detected by immunoblotting for ZBTB4 expression. Quantitative data confirmed ZBTB4 underexpression in ES tissues.
Fig. 2. The prognostic significance of ZBTB4 in ES patients. Different subgroups (ZBTB4 low/high expression) were plotted according to the cutoff values of ZBTB4, which were defined as the median of the cohort. GEO data (GSE63157 and GSE17679) revealed that ES patients with low ZBTB4 level showed a significant shorter overall survival and event-free survival compared to cases with high ZBTB4 level.
2. Materials and methods
induced ZBTB4 upregulation by targeting miRNAs inhibits tumor growth of prostate cancer in vivo [19]. Recent study figures out that loss of ZBTB4 leads to genomic instability and induces skin carcinogenesis under DMBA/TPA treatment [7]. However, there is no systematic study about the expression and role of ZBTB4 in ES. This study carried out a systematic analysis about the expression and prognostic significance of ZBTB4 based on Gene Expression Omnibus (GEO) data. Then, we investigated the role of ZBTB4 and its underlying mechanism in ES cells. We found that ZBTB4 underexpression predicts poor prognosis of ES patients. ZBTB4 inhibited proliferation of ES cells via inducing apoptosis and cell cycle arrest. Survivin might mediate the tumor suppressive role of ZBTB4 in ES. Our data possibly provide experimental and theoretical basis for ES therapeutic improvement.
2.1. ES samples, cell lines and transfection A total of 14 ES specimens were collected from patients’ resected tumor at our hospital. Inclusion criteria as follows: patients with pathological diagnosis; without radiotherapy, chemotherapy or biological treatment before operation. Four specimen of normal bones were obtained from patients treated with femoral-head replacement. All samples were immediately frozen in liquid nitrogen and stored at −80 °C. Written informed consent was obtained before collection. The study was approved by Ethics Committee of Southern Medical University. The ES cell lines, SK-ES-1 and RD-ES, were purchased from ATCC (Rockville, Maryland, USA). Cells were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and antibiotics (100 109
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Fig. 3. ZBTB4 overexpression inhibits the proliferation of ES cells. A) GEO data (GSE34620) indicated that ZBTB4 expression was inversely correlated with proliferation markers Ki-67 and PCNA levels in ES tissues. n = 117. B) SK-ES-1 and RD-ES cells that were transduced with retro-ZBTB4 or empty vector (EV) were subjected to western blotting for ZBTB4 expression. n = 3, *P < .05. C) ZBTB4 overexpression prominently reduced the proliferation capacity of ES cells. n = 3, *P < .05.
units/ml penicillin and 100 μg/ml streptomycin; Sigma-Aldrich, St. Louis, MO, USA), maintained at 37 °C in 5% CO2. Retroviral vector pMMP-ZBTB4 was generated, packaged and transduced as described [20]. Survivin expression plasmid (pcDNA3.1-survivin) and empty vector were purchased from GeneCopoeia (Guangzhou, China). ZBTB4 siRNA (sc-93593) and scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Transfection was carried out by Lipofectamine TM 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.
GCGTACAG-3′.
2.3. Western blotting Protein of cell lines, cancer and adjacent tissues was extracted with RIPA lysis buffer and its concentration was measured in accordance with BCA kit (Pierce, Rockford, IL, USA). All the samples that were quantified as 5 μg to 15 μg protein each lane were transferred into PVDF membrane after separating by 4–15% sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). After that, the membrane was sealed with 5% skimmed milk, and incubated overnight at 4 ℃ with primary antibodies including ZBTB4 (sc-514883; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Survivin (ab76424, Abcam, Cambridge, MA, USA), Ki-67 (sc-23900, Santa Cruz Biotechnology), PCNA (#13110, Cell Signaling Technology, Beverly, MA, USA), Sp1 (sc420, Santa Cruz Biotechnology), Sp3 (sc-136479, Santa Cruz Biotechnology), Sp4 (sc-390124, Santa Cruz Biotechnology) and GAPDH (sc‑47,724; Santa Cruz Biotechnology), and then incubated for 2 h at room temperature with secondary antibodies (#7076 and #7074; Cell Signaling Technology). A chemiluminescent detection system (BioRad, Hercules,CA, USA) was used for imaging.
2.2. RNA extraction and quantitative real-time PCR Total RNA of ES tissues were extracted with Trizol Reagent (Invitrogen). And cDNA was reversed with using Transcript FirststrandcDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). By using an iCycler RT-PCR system (Bio-Rad, Munich, Germany), quantitative real-time PCR could be completed by using SuperReal PreMix SYBR Green (FP204-02, TIANGEN, Beijing, China). The primers for ZBTB4 and actin were as follows: ZBTB4 forward 5′-AGGAAGTACCCC TGCCGCTA-3′, reverse 5′-TTGTAGCCTCCATTGGGTGT-3′; actin forward 5′-GGACTTCGAGCAAGAGATGG-3′, reverse 5′-AGCACTGTGTTG 110
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Fig. 4. ZBTB4 overexpression leads to apoptosis and cell cycle arrest of ES cells. A) ZBTB4 overexpression increased the portion of apoptotic ES cells. n = 3, *P < .05. B) ZBTB4 overexpression induced cell cycle arrest at G1 phase in both SK-ES-1 and RD-ES cells. n = 3, *P < .05.
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Fig. 5. ZBTB4 overexpression suppresses the tumor growth of ES in mice. SK-ES-1 cells that were transduced with retor-ZBTB4 or empty vector (EV) were subcutaneously implanted into nude mice. Tumor growth curves indicated that ZBTB4 overexpression restrained the tumor growth of ES in vivo. n = 5.
tissues based on GEO data from R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). GEO data (GSE68776) revealed that the expression ZBTB4 mRNA in ES tissues was significant lower than that in normal tissues (P = .0146, Fig. 1A). Then, investigation of our ES and normal bone tissues further confirmed underexpression of both ZBTB mRNA and protein in ES tissues compared to normal tissues (P < .0001 and P = .0017, respectively, Fig. 1B and C). The prognostic significance of ZBTB4 was disclosed using GEO data. GEO data (GSE63157 and GSE17679) suggested that ZBTB4 underexpression predicted a significant shorter overall survival and eventfree survival of ES patients (P < .05, respectively, Fig. 2). Taken together, we found that ZBTB4 was a promising prognostic biomarker for ES patients.
2.4. Cell counting kit-8 (CCK-8) assay The proliferation of ES cells was measured by CCK-8 assay. ES cells were seeded in a 96-well plates for cell culture after transfection. The specific steps as follows: each well was added with 10 μl CCK-8 reagent, fully mixed, placed in 37℃ incubator for 1 h. The optical density at 450nm of each well was measured with a Microplate reader (Bio-Rad). 2.5. Cell apoptosis assay The apoptosis of ES cells was determined by Annexin V/propidium iodide (PI) assay. After cell collection, the solution was prepared according to the instruction of Annexin V/PI method. The concentration of the cells was adjusted to 5 × 105/ml. Each tube was added with Annexin V-FITC and PI (Roche Molecular Biochemicals, Indianapolis, IN, USA), mixed and shielded from light for 10 min. Finally, the cells were added with 300 μl binding buffer and detected by flow cytometer (Beckman Coulter, Brea, CA, USA).
3.2. ZBTB4 restoration suppresses proliferation, and induces cell cycle arrest and apoptosis in ES cells
The ES cells (2 × 106 cells) that were transduced with retro-ZBTB4 or retro-EV were injected subcutaneously into the right flank of sixweek-old female nude mice. Tumor size were measured every 4 days after subcutaneous injection. Tumor volume (mm3) = longer diameter × (shorter diameter)2/2. All animal experiments were approved by the Ethics Committee of Southern Medical University.
Next, we were aimed to determine the regulatory effect of ZBTB4 on cell proliferation, cell cycle progression and apoptosis of ES cells. We first investigated the correlation between the expression of ZBTB4 and proliferation makers including Ki-67 and proliferating cell nuclear antigen (PCNA) using GEO data (GSE34620). Notably, ZBTB4 expression was inversely correlated with Ki-67 and PCNA levels in ES tissues (r = −0.480, P < .0001 and r = −0.469, P < .0001, Fig. 3A). Then, ZBTB4 was overexpressed in SK-ES-1 and RD-ES cells via retroviruses infection (P < .05, respectively, Fig. 3B). CCK-8 assay indicated that ZBTB4 restoration prominently inhibited proliferation of SK-ES-1 and RD-ES cells in vitro (P < .05, respectively, Fig. 3C). Furthermore, ZBTB4 overexpression obviously induced apoptosis of SK-ES-1 and RDES cells (P < .05, respectively, Fig. 4A). ZBTB4 restoration led to cell cycle arrest at G1 phase in both ES cell lines (P < .05, respectively, Fig. 4B). ZBTB4 overexpression increased chemosensitivity to doxorubicin, part of the regular chemotherapeutic regimen for Ewing sarcoma patients, in both SK-ES-1 and RD-ES cells. (P < .05, respectively, Supplementary Fig. 1). Thus, our data suggested that ZBTB4 prohibited cell proliferation via inducing cell cycle arrest and apoptosis in ES.
2.8. Statistical analysis
3.3. ZBTB4 restoration inhibits tumor growth of ES in mice
Measurement data were expressed as mean ± standard deviation. Statistical analysis was done by GraphPad Prism 5 software (GraphPad Software, Inc, San Diego, CA, USA) using t-test, ANOVA, Kaplan-Meier method, Log-rank test and Spearman’s rank correlation analysis, respectively. P < .05 was considered statistically significant. 3. Results
Based on our previous findings, we further invested whether ZBTB4 restoration inhibited tumor growth of ES in vivo. ZBTB4 overexpressing SK-ES-1 cells and control cells were subcutaneously implanted into nude mice, respectively. Tumor growth curves indicated that the tumor size in ZBTB overexpression group was significantly smaller than that in control group (P < .05, Fig. 5). Altogether, we found that ZBTB4 overexpression restrained the growth of ES cells in vitro and in vivo.
3.1. The expression of ZBTB4 and its prognostic significance in ES tissues
3.4. Survivin mediates the tumor suppressive role of ZBTB4 in ES cells
2.6. Cell cycle analysis Transfected ES cells were harvested and centrifuged with 1000 g for 5 min. Cells were washed with PBS and fixed with ethanol for 1 h at 4 °C. After washing with PBS for three times, cells were resuspended in 0.2 ml of RNase A buffer (1 mg/ml) at 37 °C for 30 min. Then cells were stained with 0.3 ml PI buffer (50μl/ml). The cell cycle distribution of ES cells by using the flow cytometer (FACSCalibur). 2.7. Tumor formation assay
Firstly, we investigated the expression of ES in ES and normal
Since previous studies report that ZBTB4 decreases Sp genes and 112
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Fig. 6. Survivin mediates the tumor suppressive role of ZBTB in ES cells. A) GEO data (GSE34620) indicated an inverse correlation between ZBTB4 and Survivin expression in ES tissues. n = 117. B) ZBTB4 overexpression obviously reduced Survivin abundance in both SK-ES-1 and RD-ES cells. n = 3, *P < .05. C) SK-ES-1 cells that were tranfected with indicated vectors were subjected to immunoblotting for Survivin expression. n = 3, *P < .05 vs EV group, #P < .05 vs ZBTB4 group. D) Survivin restoration enhanced the proliferation of ZBTB4 overexpressing SK-ES-1 cells. n = 3, *P < .05 vs EV group, #P < 0.05 vs ZBTB4 group. E) Survivin restoration reversed the effect of ZBTB4 overexpression on the apoptosis of SK-ES-1 cells. n = 3, *P < .05 vs EV group, #P < .05 vs ZBTB4 group. F) Survival restoration abolished the inhibitory effect of ZBTB4 overexpression on cell cycle progression in SK-ES-1 cells. n = 3, *P < .05 vs EV group, #P < .05 vs ZBTB4 group.
(Supplementary Fig. 3). While, ZBTB4 knockdown increased the expression of Survivin, Ki-67 and PCNA in RD-ES cells (Supplementary Fig. 4). Survivin is a critical regulator of cell growth and apoptosis in ES [21–23]. To clarify whether ZBTB4 plays a tumor suppressive role by targeting Survivin, Survivin expression was restored in ZBTB4 overexpressing SK-ES-1 cells via plasmid transfection (P < .05, Fig. 6C). Interestingly, Survivin restoration abolished the regulatory effect of ZBTB4 on proliferation, cell cycle progression and apoptosis in SK-ES-1 cells (P < .05, respectively, Fig. 6D–F). Taken together, ZBTB4 regulated proliferation, cell cycle progression and apoptosis at least partially
subsequent reduces Sp-related oncogenes including Survivin [9,12]. Thus, we investigated the correlation between ZBTB4 and Survivin expression in ES tissues. GEO data (GSE34620) indicated an inverse correlation between ZBTB4 and Survivin expression in ES tissues (r = −0.521, P < .0001, Fig. 6A). Then, western blotting analysis revealed that ZBTB4 overexpression led to decrease of Sp1, Sp3 and Sp4 in SK-ES-1 and RD-ES cells (Supplementary Fig. 2). Furthermore, ZBTB4 restoration reduced Survivin level in both SK-ES-1 and RD-ES cells (P < .05, respectively, Fig. 6B). And the expression of Survivin in ZBTB4 restored xenograft tissues is obviously lower than control group 113
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cycle arrest at G1 phase and apoptosis of ES cells. ZBTB4 overexpression inhibited tumor growth in vivo. Further studies show that tumor suppressive ZBTB4 reduced Survivin abundance in ES cells. Survivin at least partially mediated the tumor suppressive role of ZBTB4 in ES cells. Our findings suggested that ZBTB4 might contribute to the development of targeted therapy of ES.
through targeting Survivin in ES cells. 4. Discussion We have identified that ZBTB4 was under-expressed in ES tissues compared to normal tissues. ZBTB4 underexpression was a significant indicator for poor prognosis of ES patients. Restoration of ZBTB4 inhibited the proliferation, and induced cell cycle arrest at G1 phase and apoptosis of ES cells in vitro possibly by reducing Survivin expression. In addition, the overexpression of ZBTB4 prohibited tumor growth in vivo. Loss or underexpression of tumor suppressors is closely associated with human cancer development and progression. The aberrant expression of ZBTB4 has been shown to facilitate the progression of breast cancer [9], prostate cancer [19] and neuroblastoma [11]. Yet, the under-expression of ZBTB4 has also been spotted in skin carcinogenesis recently [7]. We found that ZBTB4 was down-regulated in ES tissues, which was consistent with GEO data involved in ES. ZBTB4 has been recognized as a novel prognostic marker for breast cancer patients [9]. Our study also confirmed that ZBTB4 underexpression contributes to shorter survival of ES patients based on two different GEO data. To date, several studies show that ZBTB4 regulates several cellular processes in human cancer. For instance, ZBTB4 restoration suppresses the capacity of breast cancer cell growth and invasion [9]. ZBTB4 is responsible for p53 induced apoptosis and cell cycle arrest via regulation of CDKN1A [8]. Drug induced ZBTB4 upregulation by targeting miRNAs inhibits tumor growth of prostate cancer in vivo [19]. Our study found that ZBTB4 expression was inversely correlated with proliferation markers, Ki-67 and PCNA. ZBTB4 knockdown increased the levels of Ki-67 and PCNA in ES cells. ZBTB4 overexpression inhibited proliferation, and led to cell cycle arrest at G1 phase and increased apoptosis of ES cells. Moreover, ZBTB4 restoration obviously slowed down the tumor growth in mice. Thus, we suggest that ZBTB4 functions as a tumor suppressor via regulating proliferation, cell cycle progression and apoptosis in ES. Recent studies report that ROS inducing anticancer agents exert treating effect via induction of the Sp transcriptional repressor ZBTB4 [13–17]. Our data showed that ZBTB4 ZBTB4 overexpression increased sensitivity to doxorubicin in ES cells. Thus, ZBTB4 may be a potential therapeutic target for ES. Survivin plays an important role in cancer initiation, development and metastasis [24,25]. Several studies have reported that Survivin overexpression is correlated with tumor grade and prognosis [26,27]. Survivin has been found to be up-regulated in a plenty of cancers including hepatocellular carcinoma, breast cancer, gastric cancer etc [28–30]. Previous studies report that ZBTB4 decreases Sp genes and subsequent reduces Sp-related oncogenes including Survivin [9,12]. Consistently, in this study, ZBTB4 was inversely correlated with Survivin expression in ES tissues. ZBTB4 overexpression reduced Sp1, Sp3, Sp4 and Survivin abundance in ES cells. And ZBTB4 knockdown increased Survivin expression in RD-ES cells. Plus, we confirmed that Survivin was a downstream mediator of ZBTB4. Taken together, ZBTB4/Survivin axis works closely during ES tumorigenesis. ZBTB4 restrains ES progression possibly by inhibiting Survivin expression. In summary, we reveal a novel function of ZBTB4 in suppressing proliferation and inducing cell cycle arrest and apoptosis of ES cells by targeting Survivin. Nevertheless, there are some limitations in this study. The underlying molecular mechanism of the down-regulation of ZBTB4 and the target relationship between ZBTB4 and Survivin in ES cells still need further investigation. Moreover, ZBTB4, as a novel biomarker and target for treatment, remains to be further explored at clinical level.
Conflicts of interest All authors declare no conflicts of interest. Acknowledgement This study was supported by the Major Projects for Industry-university-research Collaborative Innovation of Science and Technology Plan of Guangzhou (no. 201604020095), Natural Science Foundation of Guangdong Province (no. 2015A030313367), Project of Administration of Traditional Chinese Medicine of Guangdong Province (no.20161164) and 13th Five-Year the Province Project by the Guangdong Provincial Administration of Traditional Chinese Medicine Management. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2018.01.132. References [1] N. Riggi, I. Stamenkovic, The biology of Ewing sarcoma, Cancer Lett. 254 (1) (2007) 1–10. [2] E.E. Karski, K.K. Matthay, J.M. Neuhaus, R.E. Goldsby, S.G. Dubois, Characteristics and outcomes of patients with Ewing sarcoma over 40 years of age at diagnosis, Cancer Epidemiol. 37 (1) (2013) 29–33. [3] Y.S. Lau, I.E. Adamopoulos, A. Sabokbar, H. Giele, C.L. Gibbons, N.A. Athanasou, Cellular and humoral mechanisms of osteoclast formation in Ewing’s sarcoma, Br. J. Cancer 96 (11) (2007) 1716–1722. [4] N. Esiashvili, M. Goodman, R.B. Marcus Jr., Changes in incidence and survival of Ewing sarcoma patients over the past 3 decades: surveillance epidemiology and end results data, J. Pediatr. Hematol. Oncol. 30 (6) (2008) 425–430. [5] N. Gaspar, D.S. Hawkins, U. Dirksen, I.J. Lewis, S. Ferrari, M.C. Le Deley, H. Kovar, R. Grimer, J. Whelan, L. Claude, O. Delattre, M. Paulussen, P. Picci, K. Sundby Hall, H. van den Berg, R. Ladenstein, J. Michon, L. Hjorth, I. Judson, R. Luksch, M.L. Bernstein, P. Marec-Berard, B. Brennan, A.W. Craft, R.B. Womer, H. Juergens, O. Oberlin, Ewing sarcoma: current management and future approaches through collaboration, J. Clin. Oncol. 33 (27) (2015) 3036–3046. [6] Y. Jiang, J. Ludwig, F. Janku, Targeted therapies for advanced Ewing sarcoma family of tumors, Cancer Treat. Rev. 41 (5) (2015) 391–400. [7] A. Roussel-Gervais, I. Naciri, O. Kirsh, L. Kasprzyk, G. Velasco, G. Grillo, P. Dubus, P.A. Defossez, Loss of the methyl-CpG-binding protein ZBTB4 alters mitotic checkpoint, increases aneuploidy, and promotes tumorigenesis, Cancer Res. 77 (1) (2017) 62–73. [8] A. Weber, J. Marquardt, D. Elzi, N. Forster, S. Starke, A. Glaum, D. Yamada, P.A. Defossez, J. Delrow, R.N. Eisenman, H. Christiansen, M. Eilers, Zbtb4 represses transcription of P21CIP1 and controls the cellular response to p53 activation, EMBO J. 27 (11) (2008) 1563–1574. [9] K. Kim, G. Chadalapaka, S.O. Lee, D. Yamada, X. Sastre-Garau, P.A. Defossez, Y.Y. Park, J.S. Lee, S. Safe, Identification of oncogenic microRNA-17-92/ZBTB4/ specificity protein axis in breast cancer, Oncogene 31 (8) (2012) 1034–1044. [10] H. Ross-Adams, A.D. Lamb, M.J. Dunning, S. Halim, J. Lindberg, C.M. Massie, L.A. Egevad, R. Russell, A. Ramos-Montoya, S.L. Vowler, N.L. Sharma, J. Kay, H. Whitaker, J. Clark, R. Hurst, V.J. Gnanapragasam, N.C. Shah, A.Y. Warren, C.S. Cooper, A.G. Lynch, R. Stark, I.G. Mills, H. Gronberg, D.E. Neal, P.S.G. CamCa, Integration of copy number and transcriptomics provides risk stratification in prostate cancer: a discovery and validation cohort study, EBioMedicine 2 (9) (2015) 1133–1144. [11] D. Yamada, R. Perez-Torrado, G. Filion, M. Caly, B. Jammart, V. Devignot, N. Sasai, P. Ravassard, J. Mallet, X. Sastre-Garau, M.L. Schmitz, P.A. Defossez, The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4, Oncogene 28 (27) (2009) 2535–2544. [12] G. Chadalapaka, I. Jutooru, S. Safe, Celastrol decreases specificity proteins (Sp) and fibroblast growth factor receptor-3 (FGFR3) in bladder cancer cells, Carcinogenesis 33 (4) (2012) 886–894. [13] K. Karki, E. Hedrick, R. Kasiappan, U.H. Jin, S. Safe, Piperlongumine induces reactive oxygen species (ROS)-dependent downregulation of specificity protein transcription factors, Cancer Prev. Res. (Phila.) 10 (8) (2017) 467–477. [14] E. Hedrick, X. Li, S. Safe, Penfluridol represses integrin expression in breast cancer through induction of reactive oxygen species and downregulation of Sp
5. Conclusion In summary, this study revealed that ZBTB4 was significantly downregulated in ES tissues, such correlated with shorter survival of ES patients. Functionally, ZBTB4 suppressed proliferation, and induced cell 114
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transcription factors, Mol. Cancer Ther. 16 (1) (2017) 205–216. [15] I. Jutooru, A.S. Guthrie, G. Chadalapaka, S. Pathi, K. Kim, R. Burghardt, U.H. Jin, S. Safe, Mechanism of action of phenethylisothiocyanate and other reactive oxygen species-inducing anticancer agents, Mol. Cell. Biol. 34 (13) (2014) 2382–2395. [16] E. Hedrick, L. Crose, C.M. Linardic, S. Safe, Histone deacetylase inhibitors inhibit rhabdomyosarcoma by reactive oxygen species-dependent targeting of specificity protein transcription factors, Mol. Cancer Ther. 14 (9) (2015) 2143–2153. [17] R. Kasiappan, I. Jutooru, K. Karki, E. Hedrick, S. Safe, Benzyl isothiocyanate (BITC) induces reactive oxygen species-dependent repression of STAT3 protein by downregulation of specificity proteins in pancreatic cancer, J. Biol. Chem. 291 (53) (2016) 27122–27133. [18] R. Bomben, S. Gobessi, M. Dal Bo, S. Volinia, D. Marconi, E. Tissino, D. Benedetti, A. Zucchetto, D. Rossi, G. Gaidano, G. Del Poeta, L. Laurenti, D.G. Efremov, V. Gattei, The miR-17 approximately 92 family regulates the response to toll-like receptor 9 triggering of CLL cells with unmutated IGHV genes, Leukemia 26 (7) (2012) 1584–1593. [19] K. Kim, G. Chadalapaka, S.S. Pathi, U.H. Jin, J.S. Lee, Y.Y. Park, S.G. Cho, S. Chintharlapalli, S. Safe, Induction of the transcriptional repressor ZBTB4 in prostate cancer cells by drug-induced targeting of microRNA-17-92/106b-25 clusters, Mol. Cancer Ther. 11 (9) (2012) 1852–1862. [20] K. Tu, J. Li, V.K. Verma, C. Liu, D.D. Billadeau, G. Lamprecht, X. Xiang, L. Guo, R. Dhanasekaran, L.R. Roberts, V.H. Shah, N. Kang, Vasodilator-stimulated phosphoprotein promotes activation of hepatic stellate cells by regulating Rab11-dependent plasma membrane targeting of transforming growth factor beta receptors, Hepatology 61 (1) (2015) 361–374. [21] S. Shelake, U.T. Sankpal, W. Paul Bowman, M. Wise, A. Ray, R. Basha, Targeting
[22]
[23]
[24] [25] [26] [27]
[28]
[29]
[30]
115
specificity protein 1 transcription factor and survivin using tolfenamic acid for inhibiting ewing sarcoma cell growth, Invest. New Drugs 35 (2) (2017) 158–165. B. Greve, F. Sheikh-Mounessi, B. Kemper, I. Ernst, M. Gotte, H.T. Eich, Survivin, a target to modulate the radiosensitivity of Ewing’s sarcoma, Strahlenther. Onkol. 188 (11) (2012) 1038–1047. O.M. Tirado, S. Mateo-Lozano, V. Notario, Roscovitine is an effective inducer of apoptosis of Ewing’s sarcoma family tumor cells in vitro and in vivo, Cancer Res. 65 (20) (2005) 9320–9327. X. Chen, N. Duan, C. Zhang, W. Zhang, Survivin and tumorigenesis: molecular mechanisms and therapeutic strategies, J. Cancer 7 (3) (2016) 314–323. M. Mobahat, A. Narendran, K. Riabowol, Survivin as a preferential target for cancer therapy, Int. J. Mol. Sci. 15 (2) (2014) 2494–2516. P.K. Jaiswal, A. Goel, R.D. Mittal, Survivin: a molecular biomarker in cancer, Indian J. Med. Res. 141 (4) (2015) 389–397. W. Feng, D. Cai, B. Zhang, G. Lou, X. Zou, Combination of HDAC inhibitor TSA and silibinin induces cell cycle arrest and apoptosis by targeting survivin and cyclinB1/ Cdk1 in pancreatic cancer cells, Biomed. Pharmacother. 74 (2015) 257–264. B. Tang, X. Liang, F. Tang, J. Zhang, S. Zeng, S. Jin, L. Zhou, Y. Kudo, G. Qi, Expression of USP22 and survivin is an indicator of malignant behavior in hepatocellular carcinoma, Int. J. Oncol. 47 (6) (2015) 2208–2216. S. Li, L. Wang, Y. Meng, Y. Chang, J. Xu, Q. Zhang, Increased levels of LAPTM4B, VEGF and survivin are correlated with tumor progression and poor prognosis in breast cancer patients, Oncotarget 8 (25) (2017) 41282–41293. Y. Gu, S. Jin, F. Wang, Y. Hua, L. Yang, Y. Shu, Z. Zhang, R. Guo, Clinicopathological significance of PI3K, Akt and survivin expression in gastric cancer, Biomed. Pharmacother. 68 (4) (2014) 471–475.