- Email: [email protected]
β-catenin pathway
Biochemical and Biophysical Research Communications xxx (2018) 1e7
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway Limin Liu a, 1, Xingsheng Hu b, 1, Dangze Sun c, Yao Wu d, Zhanwei Zhao e, * a
Xi'an Peihua University, Peihua South Road, Chang'an District, Xi'an, China Department of Oncology, Nanchong Central Hospital, Sichuan, 637000, China c Xi'an Chest Hospital, Xi'an, Shaanxi, China d College of Information and Communication, National University of Defense Technology, Xi'an, Shaanxi, China e Department of Surgery, Navy General Hospital of PLA, Beijing, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 July 2018 Accepted 1 August 2018 Available online xxx
The incidence and mortality of hepatocellular carcinoma (HCC) is high, but the mechanisms underlying the growth and progression of HCC have not been elucidated. Recently, the ZIC family member 5 (ZIC5) is emerging as an oncogene in various types of tumors. However, its expression and biological role in HCC have not been reported. This study first demonstrated that ZIC5 was up-regulated in HCC specimens, and high ZIC5 expression indicated poor prognosis of HCC patients. In addition, over-expressed ZIC5 promoted the proliferation, migration and invasion of HCC cell lines Huh7 and HepG2 in vitro and in vivo, while ZIC5 knockdown achieved the opposite effects. Actually, ZIC5 increased the expression of genes participating in Wnt/b-catenin pathway such as b-catenin and CyclinD1. ZIC5 also promoted b-catenin to enter the nucleus of HCC cells. Furthermore, silencing b-catenin abated the promoting role of ZIC5 in HCC. Overall, this study reveals a novel mechanism of ZIC5/b-catenin that mediates the invasion and metastasis of HCC and ZIC5 serves as a novel indicator for prognosis of HCC patients. © 2018 Published by Elsevier Inc.
Keywords: Hepatocellular carcinoma ZIC5 Proliferation Cancer metastasis
1. Introduction Hepatocellular carcinoma (HCC) serves as one of the most lethal cancers worldwide [1]. Though progress has been taken in the treatment for HCC, the progression, chemo-resistance and metastasis of HCC is common, which results in the poor prognosis of HCC patients [2,3]. Thus, it is crucial to elucidate the mechanisms about the carcinogenesis and progression of HCC. However, there remains a limited understanding of the underlying mechanisms. The human zinc finger of the cerebellum (ZIC) family genes, comprised of 5 members which are vertebrate homologues of the Drosophila odd-paired (OPA) gene [4]. Recently, Zic proteins have been known to play a key role in animal development [5]. Recently, emerging evidence has indicated that ZIC5 is involved in the pathological events of various diseases, including cancer [6,7]. For example, a recent study has demonstrated that ZIC5 drives
* Corresponding author. Department of Surgery, Navy General Hospital, 6# Fu Cheng Road, Beijing, 100037, PR China. E-mail address: [email protected] (Z. Zhao). 1 These authors contributed equally to this work.
melanoma aggressiveness by PDGFD-mediated activation of FAK and STAT3 [8]. In addition, over-expression of ZIC5 promotes the proliferation of non-small cell lung cancer. Analysis of The Cancer Genome Atlas (TCGA) revealed high expression of ZIC5 in many types of tumors, such as hepatocellular carcinoma, prostate, lung cancer, colorectal, esophagus, head and neck squamous and gastric cancer [6]. However, little is known about the role of ZIC5 in most of these cancers, although it is reported that ZIC5 is a growthpromoting factor for non-small cell lung cancer [9]. ZIC5 is emerging as an oncogene and may be a potential therapeutic target for cancer treatment [9]. Furthermore, study found that ZIC5 acts as a downstream gene for Notch signaling in the induction of Xenopus neural crest [10]. ZIC5 also acts downstream of Wnt pathway and regulates zebrafish cell proliferation [11]. Whether ZIC5 plays an important role of regulating the Wnt pathway in HCC remains unclear. The Wnt/b-catenin pathway has been reported to be crucial in regulating development, carcinogenesis and stem cell self-renewal [12,13]. b-catenin, an effector in Wnt/b-catenin pathway, In the “signal off” state, b-catenin binds to the complex of Axin and APC in the cytoplasm; then, GSK3b and CKIa induce phosphorylation degradation of b-catenin via the ubiquitin-proteasome system.
https://doi.org/10.1016/j.bbrc.2018.08.009 0006-291X/© 2018 Published by Elsevier Inc.
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
2
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
However, b-catenin enters the nucleus when activated and functions as an activator to trigger the expression of downstream genes in the “signal on” state that promote tumor growth and progression [14e16]. Additionally, b-catenin signaling is activated in HCC and promoted carcinogenesis and metastasis of HCC [17], but the upstream regulators of Wnt/b-catenin in HCC have not been elucidated well. In our study, high ZIC5 expression was observed in most HCC specimens and indicated poor prognosis of HCC patients. We also found that over-expressed ZIC5 promoted the proliferation, migration and invasion of HCC cells in vitro and in vivo, while silencing ZIC5 expression gained the opposite effects. Furthermore, ZIC5 increased the expression of b-catenin and CyclinD1, and promoted b-catenin to enter the nucleus of HCC cells. In addition, silencing b-catenin abated the promoting role of ZIC5 in HCC cells. Our findings indicate that ZIC5 serves as a novel indicator for prognosis of HCC patients and may be a potential therapeutic target for HCC treatment.
2.2. Immunohistochemistry
2. Materials and methods
2.4. Gene knockdown and plasmid transfection
2.1. Patients and specimens
The HCC cell lines were respectively transfected with the shRNAs expressing lentivirus (shZIC5 or shb-catenin) or control lentivirus. After 72h transfection, the cells were observed and photographed under microscope. The sequences for shRNA were presented in Supplementary Table S1. ZIC5 plasmid transfection was carried out using Lipofectamine 3000 reagents (Invitrogen) according to the manufacturer's protocols. The sequence of ZIC5 plasmid was shown in Supplementary Table S1.
The samples were obtained after writing informed consent from patients according to an established protocol approved by the Ethics Committee in our Hospital. Patients were pathologically diagnosed HCC in our Hospital. The clinicopathological features of patients were summarized in Table 1. All the patients were followed until 2013, with a median observation time of 54 months.
HCC tissue slides were stained with the following primary antibodies at 4 C overnight: anti-ZIC5 (Abcam, USA) and anti-b-catenin (Abcam). The secondary antibodies and diaminobenzidine (DAB) reagent (Dako, CA) were employed in the detection procedure. All slides were observed and photographed with an Olympus microscope (Japan). The staining levels in all clinical samples were examined by two independent pathologists. Low expression level was defined as score 0 or 1, whereas high expression in tumor tissues was identified as score 2 or 3. 2.3. Cell lines and cell culture HCC cell lines Huh7 and HepG2 were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (China). HCC cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco).
2.5. Cell proliferation assay and Western blot Table 1 Summary of clinicopathologic variables. Characteristic
No. of patients
Patient Sex Male Female Age (year) childpugh Yes No AFP (ng/ml)a 400 < 400 Tumor size (cm) Tumor number (count) Tumor differentiation grade 2 3 4 Grade 2 3 LC Yes No Tumor satellites Yes No Recurrence Yes No Expired Yes No Risk-free Survival time (mo) Time of folow-up (mo) a
AFP, serum -fetoprotein.
67 2 34 - 73 (median, 51)
The cell proliferation was examined by a CCK-8 (CK-04, Japan) according to the manufacturer's protocols. The protein of HCC cells were extracted using the NE-PER nuclear and cytoplasmic extraction kit (USA). Primary antibodies as follows were used, anti-ZIC5, b-catenin and anti-b-actin from Abcam. Secondary antibodies were goat anti-rabbit IgG-HRP-linked antibody from Cell Signaling Technology (USA).
60 9
2.6. Animal experiments
41 28 1.5e13 (median, 5) 1 - 4 (median, 1)
The Huh7 cells were transfected with the luciferase reporter gene, and the cells from different treatments were respectively injected into six-week-old male NOD-SCID mice. Tumor growth was monitored weekly by live-animal bioluminescence optical imaging with the IVIS Lumina II imaging system (USA) after intraperitoneal injecting of D-luciferin (150 mg/kg) (Gold Biotech, USA) in PBS. Mice were sacrificed 6 weeks after tumor implantation. All experimental animal procedures were approved by the Animal Care and Use Committee.
3 65 1 68 1 53 16 57 12 51 18 39 30 1 - 75 (median, 12) 1 - 90 (median, 40)
2.7. Wound healing and invasion assays Wound-healing assay and invasion assay were performed as described previously [18]. Cell migration was assessed by measuring the movement of the cells into the scraper. After the cells were covered with the bottom of the plate, 200 ml pipette was used to create cellular area. The spread of wound closure was observed after 24 h and photographed under a microscope. The % wound healing was quantified by the space of migration tumor cells at 24 h after scrambled/the space of wound at 0 h 100%. The amount of cell invasion was measured in vitro using a Transwell chamber containing a Matrigel-coated polycarbonate membrane
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
3
Fig. 1. High ZIC5 expression in specimens indicates poor prognosis of HCC patients. A, Representative immunohistochemistry (IHC) staining of ZIC5 in human HCC specimens are shown (scale bar ¼ 50 mm). B-C, Kaplan-Meier curves for the overall survival (OS) and recurrence-free survival (RFS) of patients with HCC were analyzed according to ZIC5 expression in HCC specimens (n ¼ 69).
filter (8-mm pore size)(Sigma, St. Louis, USA). Briefly, 48 h after transfection, cells were washed twice using DMEM and triplicated in a Transwell chamber containing 200 ml of serum-free medium. About 700 ml of medium containing 10% fetal bovine serum was added to the lower chamber. The plates were incubated for 24 h at 37 C. The cells that had invaded into the bottom surface of the filter were fixed with methanol and stained with hematoxylin. The invasive ability was determined by counting the penetrating cells under a microscope at 200 magnification on 10 random fields in each well. The experiments were performed in triplicate. 2.8. Statistical analysis Numerical data were expressed as the mean ± S.D. Statistical differences between variables were analyzed by two-tailed Student's t-test, chi-square test or Fisher's exact test for categorical/
binary measures and ANOVA for continuous measures. Survival curve was plotted by the Kaplan-Meier method and compared using the log-rank analysis. Difference was considered significant at P < 0.05. Data analysis was performed by using SPSS software. 3. Results 3.1. High ZIC5 expression in specimens indicates poor prognosis of HCC patients To investigate the relationship between ZIC5 expression and the clinicopathological features and prognosis of HCC patients. First, ZIC5 expression was examined in HCC tissues by immunohistochemistry (IHC) assays (Fig. 1A). According to ZIC5 expression in specimens, patients were divided into ZIC5low group (n ¼ 28) and ZIC5high group (n ¼ 41) (Table 2). ZIC5 expression associated with
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
4
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
Table 2 Clinicopathologic Characteristics of liver cancer Subtypes Defined by ZIC5 Expression. liver cancer subtypes
Sex Male Female Age (year)b childpugh Yes No AFP (ng/ml) 400 < 400 Tumor size (cm)b Tumor number (count)b Tumor differentiation grade 2 3 4 grade 2 3 LC Yes No Tumor satellites Yes No Recurrence Yes No Expired Yes No Reference-free survival time (mo)b Overall survival time (mo)b
ZIC5 expression Low (n ¼ 28)
High (n ¼ 41)
P valuea
28 0 51.96 ± 2.09
39 2 51.02 ± 1.59
0.511
24 4
36 5
1.000
11 17 4.45 ± 0.41 1.11 ± 0.06
17 24 6.67 ± 0.46 1.37 ± 0.12
1.000
2 25 1
1 40 0
0.296
27 1
41 0
0.406
19 9
34 7
0.160
22 6
35 6
0.527
15 13
3 38
<0.001
22 6 48.39 ± 4.75 59.29 ± 4.10
8 33 14.15 ± 3.19 28.68 ± 3.85
<0.001
0.718
0.001 0.096
<0.001 <0.001
a Statistical significance was caluculated by chi-square test or fisher's exact test for categorical/binary measures and ANOVA for continuous measures. b Data are presented as mean ± SE.
tumor size (p ¼ 0.004) and tumor number (p ¼ 0.026) (Table 2). Furthermore, Kaplan-Meier analysis revealed that patients with high ZIC5 expression indicated worst overall survival (OS) and recurrence-free survival (RFS) (Fig. 1BeC; Table 2). The results indicate that ZIC5 serves as a novel indicator for the prognosis of HCC patients.
3.2. ZIC5 was required in proliferation and tumorigenicity of HCC We next examined whether ZIC5 mediated the growth of HCC. First, we performed ZIC5 knockdown (treated with ZIC5 shRNA) assays in HCC cell lines Huh7 or HepG2. After successful knockdown of ZIC5 validated by Western blot (Fig. 2A), the proliferation of Huh7 or HepG2 cells were detected using CCK-8. As expected, silencing ZIC5 decreased proliferation of Huh7 or HepG2 cells (Fig. 2B). Furthermore, ZIC5 knockdown and control Huh7 cells labeled with stable luciferase were subcutaneously injected into the mice, and the bioluminescence was detected using an in vivo imaging system to monitor tumor growth (Fig. 2C). As expected, ZIC5 knockdown decreased tumor incidence and the growth of Huh7 cells-derived tumor xenografts compared with the control cells (Fig. 2C). The results indicate that ZIC5 is required in growth of HCC. Matrigel invasion assays and scratch wound healing assays were tested to evaluate whether ZIC5 was responsible for the migratory and invasive capabilities of sh-ZIC5 1#-Huh7 and sh-ZIC5 1#-HepG2 cells. The Matrigel invasion assay showed that invasiveness was markedly decreased in the sh-ZIC5 1#-treated cells
Fig. 2. ZIC5 was required in proliferation, invasion, migration and tumorigenicity of HCC. A, The expression of ZIC5 in Huh7 or HepG2 cells treated with ZIC5 shRNAs or control shRNA was examined by Western blot. B, CCK-8 experiments were employed to detect the proliferation of Huh7 or HepG2 cells with ZIC5 shRNAs or control shRNA at the indicated times. C, Huh7 cells treated with ZIC5 shRNAs or control shRNA were respectively subcutaneously injected into mice (n ¼ 6/group), and the tumor incidence and growth was evaluated by bioluminescent imaging. D, The invasive properties of the cells were analyzed by an invasion assay using a Matrigel-coated Boyden chamber. E, The cell migration rates of ZIC5 shRNA-Huh7 and ZIC5 shRNA-HepG2 were tested by a wound-healing assay. Microscopic observation of wound closure was performed 0 and 24 h after scratching the cell layer. (*p < 0.05, **p < 0.01 and ***p < 0.001).
compared with the empty vector-transfected cells (sh-NC) (p < 0.05, Fig. 2D). Also, sh-ZIC5 1# decreased cell motility in HepG2 cells (Fig. 2E).
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
5
Fig. 3. ZIC5 promotes proliferation, invasion, migration and tumorigenicity of HCC cell. A, The expression of ZIC5 in Huh7 or HepG2 cells without or with ZIC5 over-expression (OE) was performed by Western blot. B, CCK-8 experiments were used to detect the proliferation of Huh7 or HepG2 cells without or with ZIC5 over-expression at the indicated times. C, Huh7 cells without or with ZIC5 over-expression were respectively subcutaneously injected into mice (n ¼ 6/group). The tumor growth was evaluated by bioluminescent imaging. D, The invasive properties of the cells were analyzed by an invasion assay using a Matrigel-coated Boyden chamber. E, The cell migration rates of ZIC5 overexpression (OE) (pcDNAZIC5) and control (Con) (pcDNA3.1 control vectors) were tested by a wound-healing assay. Microscopic observation of wound closure was performed 0 and 24 h after scratching the cell layer. (*p < 0.05, **p < 0.01 and ***p < 0.001).
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
6
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
3.3. ZIC5 promotes proliferation and tumorigenicity of HCC cells We next examined whether enforced ZIC5 facilitated the growth of HCC cells. First, ZIC5 was over-expressed (OE) in Huh7 or HepG2 cells by pcDNA-ZIC5, which was validated by Western blot (Fig. 3A). Then CCK-8 assays demonstrated that ZIC5 facilitated the proliferation of HCC cells (Fig. 3B). Second, ZIC5 over-expressed and control Huh7 cells labeled with stable luciferase were subcutaneously injected into the mice. The bioluminescence was detected using an in vivo imaging system to monitor tumor growth (Fig. 3C). As expected, ZIC5 promoted the growth of tumors derived from Huh7 cells (Fig. 3C). Compared with the empty vector-transfected cells, overexpression of ZIC5 increased cell invasiveness and migration by the matrigel invasion assay and scratch wound healing assay (p < 0.05, Fig. 3D and E). 3.4. ZIC5 promoted growth of HCC in a b-catenin dependant manner It is well-known that activated Wnt/b-catenin pathway facilitates the growth and progression of HCC. Then we determined whether ZIC5 regulatedb-catenin signaling in HCC cells. First, overexpressed ZIC5 increased the protein expression of b-catenin and CyclinD1 in Huh7 or HepG2 cells (Fig. 4A), while silencing ZIC5 decreased the b-catenin and CyclinD1 expressions in HCC cell lines
(Fig. 4B). Second, immunofluorescence assays demonstrated that over-expressed ZIC5 promptedb-catenin to enter the nucleus of HCC cells (Fig. 4C). Thus, ZIC5 activated b-catenin signaling in HCC. Furthermore, we determined whether b-catenin was required for the role of ZIC5 in HCC cell lines. CCK-8 assays presented that bcatenin knockdown alleviated the pro-tumor effects of ZIC5 in HCC cells (Fig. 4D). The results indicate that ZIC5 promotes the growth and progression of HCC via b-catenin. 4. Discussion ZIC5 has been reported to be implicated in various tumors, but there is not a literature reporting its role in HCC. Our study was the first to elucidate the expression and significance of ZIC5 in human HCC specimens, and elucidate its promoting role in growth and progression of HCC. To determine the significance of ZIC5 in patients with HCC, we first detected its expression in HCC specimens by IHC staining. Our results demonstrated that most of the HCC tissues exhibited high ZIC5 expression. Furthermore, high ZIC5 expression indicated worse overall survival and disease-free survival of HCC patients, which prompted us to examine whether ZIC5 exerted a pro-tumor function in growth of HCC. As expected, overexpressed or silencing ZIC5 respectively increased or inhibited the proliferation, migration and invasion of HCC cells in vitro and in vivo.
Fig. 4. ZIC5 promoted growth and progression of HCC in a b-catenin dependant manner. A, Western blot analysis was performed to detectb-catenin and CyclinD1 expressions in Huh7 or HepG2 cells with ZIC5 over-expression or control. B, b-catenin and CyclinD1 expressions in Huh7 or HepG2 cells with ZIC5 shRNAs or control shRNA was performed by Western blot analysis. C, Cell immunofluorescence was use to examine the location of b-catenin in the nucleus and cytoplasm of Huh7 or HepG2 cells with ZIC5 over-expression or control (scar bar ¼ 10 mm). D, CCK-8 experiments were used to assess the proliferation of Huh7 or HepG2 cells without or with ZIC5 over-expression, and with ZIC5 over-expression plus b-catenin knockdown (sh-b-catenin) at the indicated times. (*p < 0.05, **p < 0.01 and ***p < 0.001).
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
Additionally, we revealed a novel mechanism underlying ZIC5 in activating b-catenin in HCC cells. Though previous study showed the b-catenin facilitated the growth and progression of in HCC [19,20], whether ZIC5 mediated b-catenin in HCC has not been elucidated. Our study indicated that b-catenin was necessary in ZIC5-regulaitng growth of HCC. Thus, ZIC5 may be served as a crucial indicator for prognosis of HCC patients and a novel target for HCC treatment. Our studies will examine how ZIC5 regulated bcatenin in HCC cells and evaluate the effect of targeting ZIC5 and related pathways on HCC treatment in vivo. CyclinD1 is cell-cycle proteins. We only tested the expression of cyclin D1 after silencing or overexpressing ZIC5. It show that CyclinD1 was regulated by ZIC5 in HCC. However, it was not known which mechanism was involved in the proliferation of HCC by ZIC5 regulation or whether it was related to the cell cycle. This is what we will continue to study later. Conflicts of interest The authors declare that they have no competing interests. Acknowledgments This work was supported by The National Natural Science Foundation of China (81400699) Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.08.009. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.08.009. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, Ca - Cancer J. Clin. 65 (2015) (2012) 87e108. [2] X. Sun, S.C. Wang, Y. Wei, X. Luo, Y. Jia, L. Li, P. Gopal, M. Zhu, I. Nassour, J.C. Chuang, T. Maples, C. Celen, L.H. Nguyen, L. Wu, S. Fu, W. Li, L. Hui, F. Tian, Y. Ji, S. Zhang, M. Sorouri, T.H. Hwang, L. Letzig, L. James, Z. Wang, A.C. Yopp, A.G. Singal, H. Zhu, Arid1a has context-dependent oncogenic and tumor suppressor functions in liver cancer, Canc. Cell 32 (2017) 574e589, e576. [3] C. Wang, S.Y. Fu, M.D. Wang, W.B. Yu, Q.S. Cui, H.R. Wang, H. Huang, W. Dong, W.W. Zhang, P.P. Li, C. Lin, Z.Y. Pan, Y. Yang, M.C. Wu, W.P. Zhou, Zinc finger protein X-linked promotes expansion of EpCAM(þ) cancer stem-like cells in hepatocellular carcinoma, Mol. Oncol. 11 (2017) 455e469.
7
[4] G. Ma, W. Dai, A. Sang, X. Yang, Q. Li, Roles of ZIC family genes in human gastric cancer, Int. J. Mol. Med. 38 (2016) 259e266. [5] J. Aruga, The role of Zic genes in neural development, Mol. Cell. Neurosci. 26 (2004) 205e221. [6] R. Satow, S. Inagaki, C. Kato, M. Shimozawa, K. Fukami, Identification of zinc finger protein of the cerebellum 5 as a survival factor of prostate and colorectal cancer cells, Canc. Sci. 108 (2017) 2405e2412. [7] W. Han, C. Zhang, X.J. Gao, H.B. Wang, F. Chen, F. Cao, Y.W. Hu, J. Ma, X. Gu, H.Z. Ding, Clinicopathologic and prognostic significance of the zinc finger of the cerebellum family in invasive breast cancer, J. Breast Canc. 21 (2018) 51e61. [8] R. Satow, T. Nakamura, C. Kato, M. Endo, M. Tamura, R. Batori, S. Tomura, Y. Murayama, K. Fukami, ZIC5 drives melanoma aggressiveness by PDGFDmediated activation of FAK and STAT3, Canc. Res. 77 (2017) 366e377. [9] Q. Sun, R. Shi, X. Wang, D. Li, H. Wu, B. Ren, Overexpression of ZIC5 promotes proliferation in non-small cell lung cancer, Biochem. Biophys. Res. Commun. 479 (2016) 502e509. [10] Y. Shi, J. Li, C. Chen, Y. Xia, Y. Li, P. Zhang, Y. Xu, T. Li, W. Zhou, W. Song, Ketamine modulates Zic5 expression via the Notch signaling pathway in neural crest induction, Front. Mol. Neurosci. 11 (2018) 9. [11] M.K. Nyholm, S.F. Wu, R.I. Dorsky, Y. Grinblat, The zebrafish zic2a-zic5 gene pair acts downstream of canonical Wnt signaling to control cell proliferation in the developing tectum, Development 134 (2007) 735e746. [12] H.K. Lee, E.W. Lee, J. Seo, M. Jeong, S.H. Lee, S.Y. Kim, E.H. Jho, C.H. Choi, J.Y. Chung, J. Song, Ubiquitylation and degradation of adenomatous polyposis coli by MKRN1 enhances Wnt/beta-catenin signaling, Oncogene (2018 May 1), https://doi.org/10.1038/s41388-018-0267-3 [Epub ahead of print]. [13] C. Wang, M.D. Wang, P. Cheng, H. Huang, W. Dong, W.W. Zhang, P.P. Li, C. Lin, Z.Y. Pan, M.C. Wu, W.P. Zhou, Hepatitis B virus X protein promotes the stemlike properties of OV6(þ) cancer cells in hepatocellular carcinoma, Cell Death Dis. 8 (2017), e2560. [14] X. Jiang, P.Y. Mak, H. Mu, W. Tao, D.H. Mak, S. Kornblau, Q. Zhang, P. Ruvolo, J.K. Burks, W. Zhang, T. McQueen, R. Pan, H. Zhou, M. Konopleva, J. Cortes, Q. Liu, M. Andreeff, B.Z. Carter, Disruption of Wnt/beta-catenin exerts antileukemia activity and synergizes with FLT3 inhibition in FLT3-mutant acute myeloid leukemia, Clin. Canc. Res. Off. J. Am. Assoc. Canc. Res. 24 (2018) 2417e2429. [15] Q. Chen, J. Cai, Q. Wang, Y. Wang, M. Liu, J. Yang, J. Zhou, C. Kang, M. Li, C. Jiang, Long noncoding RNA NEAT1, regulated by the EGFR pathway, contributes to glioblastoma progression through the WNT/beta-Catenin pathway by scaffolding EZH2, Clin. Canc. Res. Off. J. Am. Assoc. Canc. Res. 24 (2018) 684e695. [16] H. Clevers, Wnt/beta-catenin signaling in development and disease, Cell 127 (2006) 469e480. [17] S. Fatima, X. Shi, Z. Lin, G.Q. Chen, X.H. Pan, J.C. Wu, J.W. Ho, N.P. Lee, H. Gao, G. Zhang, A. Lu, Z.X. Bian, 5-Hydroxytryptamine promotes hepatocellular carcinoma proliferation by influencing beta-catenin, Mol Oncol 10 (2016) 195e212. [18] B. Zhou, H. Chen, D. Wei, Y. Kuang, X. Zhao, G. Li, J. Xie, P. Chen, A novel miR219-SMC4-JAK2/Stat3 regulatory pathway in human hepatocellular carcinoma, J. Exp. Clin. Canc. Res. 33 (2014) 55. [19] H. Yin, R. Que, C. Liu, W. Ji, B. Sun, X. Lin, Q. Zhang, X. Zhao, Z. Peng, X. Zhang, H. Qian, L. Chen, Y. Yao, C. Su, Survivin-targeted drug screening platform identifies a matrine derivative WM-127 as a potential therapeutics against hepatocellular carcinoma, Canc. Lett. 425 (2018) 54e64. [20] Y.F. Sun, W. Guo, Y. Xu, Y.H. Shi, Z.J. Gong, Y. Ji, M. Du, X. Zhang, B. Hu, A. Huang, G.G. Chen, P.B.S. Lai, Y. Cao, S.J. Qiu, J. Zhou, X.R. Yang, J. Fan, Circulating tumor cells from different vascular sites exhibit spatial heterogeneity in epithelial and mesenchymal composition and distinct clinical significance in hepatocellular carcinoma, Clin. Canc. Res. Off. J. Am. Assoc. Canc. Res. 24 (2018) 547e559.
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway Limin Liu a, 1, Xingsheng Hu b, 1, Dangze Sun c, Yao Wu d, Zhanwei Zhao e, * a
Xi'an Peihua University, Peihua South Road, Chang'an District, Xi'an, China Department of Oncology, Nanchong Central Hospital, Sichuan, 637000, China c Xi'an Chest Hospital, Xi'an, Shaanxi, China d College of Information and Communication, National University of Defense Technology, Xi'an, Shaanxi, China e Department of Surgery, Navy General Hospital of PLA, Beijing, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 July 2018 Accepted 1 August 2018 Available online xxx
The incidence and mortality of hepatocellular carcinoma (HCC) is high, but the mechanisms underlying the growth and progression of HCC have not been elucidated. Recently, the ZIC family member 5 (ZIC5) is emerging as an oncogene in various types of tumors. However, its expression and biological role in HCC have not been reported. This study first demonstrated that ZIC5 was up-regulated in HCC specimens, and high ZIC5 expression indicated poor prognosis of HCC patients. In addition, over-expressed ZIC5 promoted the proliferation, migration and invasion of HCC cell lines Huh7 and HepG2 in vitro and in vivo, while ZIC5 knockdown achieved the opposite effects. Actually, ZIC5 increased the expression of genes participating in Wnt/b-catenin pathway such as b-catenin and CyclinD1. ZIC5 also promoted b-catenin to enter the nucleus of HCC cells. Furthermore, silencing b-catenin abated the promoting role of ZIC5 in HCC. Overall, this study reveals a novel mechanism of ZIC5/b-catenin that mediates the invasion and metastasis of HCC and ZIC5 serves as a novel indicator for prognosis of HCC patients. © 2018 Published by Elsevier Inc.
Keywords: Hepatocellular carcinoma ZIC5 Proliferation Cancer metastasis
1. Introduction Hepatocellular carcinoma (HCC) serves as one of the most lethal cancers worldwide [1]. Though progress has been taken in the treatment for HCC, the progression, chemo-resistance and metastasis of HCC is common, which results in the poor prognosis of HCC patients [2,3]. Thus, it is crucial to elucidate the mechanisms about the carcinogenesis and progression of HCC. However, there remains a limited understanding of the underlying mechanisms. The human zinc finger of the cerebellum (ZIC) family genes, comprised of 5 members which are vertebrate homologues of the Drosophila odd-paired (OPA) gene [4]. Recently, Zic proteins have been known to play a key role in animal development [5]. Recently, emerging evidence has indicated that ZIC5 is involved in the pathological events of various diseases, including cancer [6,7]. For example, a recent study has demonstrated that ZIC5 drives
* Corresponding author. Department of Surgery, Navy General Hospital, 6# Fu Cheng Road, Beijing, 100037, PR China. E-mail address: [email protected] (Z. Zhao). 1 These authors contributed equally to this work.
melanoma aggressiveness by PDGFD-mediated activation of FAK and STAT3 [8]. In addition, over-expression of ZIC5 promotes the proliferation of non-small cell lung cancer. Analysis of The Cancer Genome Atlas (TCGA) revealed high expression of ZIC5 in many types of tumors, such as hepatocellular carcinoma, prostate, lung cancer, colorectal, esophagus, head and neck squamous and gastric cancer [6]. However, little is known about the role of ZIC5 in most of these cancers, although it is reported that ZIC5 is a growthpromoting factor for non-small cell lung cancer [9]. ZIC5 is emerging as an oncogene and may be a potential therapeutic target for cancer treatment [9]. Furthermore, study found that ZIC5 acts as a downstream gene for Notch signaling in the induction of Xenopus neural crest [10]. ZIC5 also acts downstream of Wnt pathway and regulates zebrafish cell proliferation [11]. Whether ZIC5 plays an important role of regulating the Wnt pathway in HCC remains unclear. The Wnt/b-catenin pathway has been reported to be crucial in regulating development, carcinogenesis and stem cell self-renewal [12,13]. b-catenin, an effector in Wnt/b-catenin pathway, In the “signal off” state, b-catenin binds to the complex of Axin and APC in the cytoplasm; then, GSK3b and CKIa induce phosphorylation degradation of b-catenin via the ubiquitin-proteasome system.
https://doi.org/10.1016/j.bbrc.2018.08.009 0006-291X/© 2018 Published by Elsevier Inc.
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
2
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
However, b-catenin enters the nucleus when activated and functions as an activator to trigger the expression of downstream genes in the “signal on” state that promote tumor growth and progression [14e16]. Additionally, b-catenin signaling is activated in HCC and promoted carcinogenesis and metastasis of HCC [17], but the upstream regulators of Wnt/b-catenin in HCC have not been elucidated well. In our study, high ZIC5 expression was observed in most HCC specimens and indicated poor prognosis of HCC patients. We also found that over-expressed ZIC5 promoted the proliferation, migration and invasion of HCC cells in vitro and in vivo, while silencing ZIC5 expression gained the opposite effects. Furthermore, ZIC5 increased the expression of b-catenin and CyclinD1, and promoted b-catenin to enter the nucleus of HCC cells. In addition, silencing b-catenin abated the promoting role of ZIC5 in HCC cells. Our findings indicate that ZIC5 serves as a novel indicator for prognosis of HCC patients and may be a potential therapeutic target for HCC treatment.
2.2. Immunohistochemistry
2. Materials and methods
2.4. Gene knockdown and plasmid transfection
2.1. Patients and specimens
The HCC cell lines were respectively transfected with the shRNAs expressing lentivirus (shZIC5 or shb-catenin) or control lentivirus. After 72h transfection, the cells were observed and photographed under microscope. The sequences for shRNA were presented in Supplementary Table S1. ZIC5 plasmid transfection was carried out using Lipofectamine 3000 reagents (Invitrogen) according to the manufacturer's protocols. The sequence of ZIC5 plasmid was shown in Supplementary Table S1.
The samples were obtained after writing informed consent from patients according to an established protocol approved by the Ethics Committee in our Hospital. Patients were pathologically diagnosed HCC in our Hospital. The clinicopathological features of patients were summarized in Table 1. All the patients were followed until 2013, with a median observation time of 54 months.
HCC tissue slides were stained with the following primary antibodies at 4 C overnight: anti-ZIC5 (Abcam, USA) and anti-b-catenin (Abcam). The secondary antibodies and diaminobenzidine (DAB) reagent (Dako, CA) were employed in the detection procedure. All slides were observed and photographed with an Olympus microscope (Japan). The staining levels in all clinical samples were examined by two independent pathologists. Low expression level was defined as score 0 or 1, whereas high expression in tumor tissues was identified as score 2 or 3. 2.3. Cell lines and cell culture HCC cell lines Huh7 and HepG2 were purchased from Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (China). HCC cell lines were cultured in Dulbecco's modified Eagle's medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco).
2.5. Cell proliferation assay and Western blot Table 1 Summary of clinicopathologic variables. Characteristic
No. of patients
Patient Sex Male Female Age (year) childpugh Yes No AFP (ng/ml)a 400 < 400 Tumor size (cm) Tumor number (count) Tumor differentiation grade 2 3 4 Grade 2 3 LC Yes No Tumor satellites Yes No Recurrence Yes No Expired Yes No Risk-free Survival time (mo) Time of folow-up (mo) a
AFP, serum -fetoprotein.
67 2 34 - 73 (median, 51)
The cell proliferation was examined by a CCK-8 (CK-04, Japan) according to the manufacturer's protocols. The protein of HCC cells were extracted using the NE-PER nuclear and cytoplasmic extraction kit (USA). Primary antibodies as follows were used, anti-ZIC5, b-catenin and anti-b-actin from Abcam. Secondary antibodies were goat anti-rabbit IgG-HRP-linked antibody from Cell Signaling Technology (USA).
60 9
2.6. Animal experiments
41 28 1.5e13 (median, 5) 1 - 4 (median, 1)
The Huh7 cells were transfected with the luciferase reporter gene, and the cells from different treatments were respectively injected into six-week-old male NOD-SCID mice. Tumor growth was monitored weekly by live-animal bioluminescence optical imaging with the IVIS Lumina II imaging system (USA) after intraperitoneal injecting of D-luciferin (150 mg/kg) (Gold Biotech, USA) in PBS. Mice were sacrificed 6 weeks after tumor implantation. All experimental animal procedures were approved by the Animal Care and Use Committee.
3 65 1 68 1 53 16 57 12 51 18 39 30 1 - 75 (median, 12) 1 - 90 (median, 40)
2.7. Wound healing and invasion assays Wound-healing assay and invasion assay were performed as described previously [18]. Cell migration was assessed by measuring the movement of the cells into the scraper. After the cells were covered with the bottom of the plate, 200 ml pipette was used to create cellular area. The spread of wound closure was observed after 24 h and photographed under a microscope. The % wound healing was quantified by the space of migration tumor cells at 24 h after scrambled/the space of wound at 0 h 100%. The amount of cell invasion was measured in vitro using a Transwell chamber containing a Matrigel-coated polycarbonate membrane
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
3
Fig. 1. High ZIC5 expression in specimens indicates poor prognosis of HCC patients. A, Representative immunohistochemistry (IHC) staining of ZIC5 in human HCC specimens are shown (scale bar ¼ 50 mm). B-C, Kaplan-Meier curves for the overall survival (OS) and recurrence-free survival (RFS) of patients with HCC were analyzed according to ZIC5 expression in HCC specimens (n ¼ 69).
filter (8-mm pore size)(Sigma, St. Louis, USA). Briefly, 48 h after transfection, cells were washed twice using DMEM and triplicated in a Transwell chamber containing 200 ml of serum-free medium. About 700 ml of medium containing 10% fetal bovine serum was added to the lower chamber. The plates were incubated for 24 h at 37 C. The cells that had invaded into the bottom surface of the filter were fixed with methanol and stained with hematoxylin. The invasive ability was determined by counting the penetrating cells under a microscope at 200 magnification on 10 random fields in each well. The experiments were performed in triplicate. 2.8. Statistical analysis Numerical data were expressed as the mean ± S.D. Statistical differences between variables were analyzed by two-tailed Student's t-test, chi-square test or Fisher's exact test for categorical/
binary measures and ANOVA for continuous measures. Survival curve was plotted by the Kaplan-Meier method and compared using the log-rank analysis. Difference was considered significant at P < 0.05. Data analysis was performed by using SPSS software. 3. Results 3.1. High ZIC5 expression in specimens indicates poor prognosis of HCC patients To investigate the relationship between ZIC5 expression and the clinicopathological features and prognosis of HCC patients. First, ZIC5 expression was examined in HCC tissues by immunohistochemistry (IHC) assays (Fig. 1A). According to ZIC5 expression in specimens, patients were divided into ZIC5low group (n ¼ 28) and ZIC5high group (n ¼ 41) (Table 2). ZIC5 expression associated with
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
4
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
Table 2 Clinicopathologic Characteristics of liver cancer Subtypes Defined by ZIC5 Expression. liver cancer subtypes
Sex Male Female Age (year)b childpugh Yes No AFP (ng/ml) 400 < 400 Tumor size (cm)b Tumor number (count)b Tumor differentiation grade 2 3 4 grade 2 3 LC Yes No Tumor satellites Yes No Recurrence Yes No Expired Yes No Reference-free survival time (mo)b Overall survival time (mo)b
ZIC5 expression Low (n ¼ 28)
High (n ¼ 41)
P valuea
28 0 51.96 ± 2.09
39 2 51.02 ± 1.59
0.511
24 4
36 5
1.000
11 17 4.45 ± 0.41 1.11 ± 0.06
17 24 6.67 ± 0.46 1.37 ± 0.12
1.000
2 25 1
1 40 0
0.296
27 1
41 0
0.406
19 9
34 7
0.160
22 6
35 6
0.527
15 13
3 38
<0.001
22 6 48.39 ± 4.75 59.29 ± 4.10
8 33 14.15 ± 3.19 28.68 ± 3.85
<0.001
0.718
0.001 0.096
<0.001 <0.001
a Statistical significance was caluculated by chi-square test or fisher's exact test for categorical/binary measures and ANOVA for continuous measures. b Data are presented as mean ± SE.
tumor size (p ¼ 0.004) and tumor number (p ¼ 0.026) (Table 2). Furthermore, Kaplan-Meier analysis revealed that patients with high ZIC5 expression indicated worst overall survival (OS) and recurrence-free survival (RFS) (Fig. 1BeC; Table 2). The results indicate that ZIC5 serves as a novel indicator for the prognosis of HCC patients.
3.2. ZIC5 was required in proliferation and tumorigenicity of HCC We next examined whether ZIC5 mediated the growth of HCC. First, we performed ZIC5 knockdown (treated with ZIC5 shRNA) assays in HCC cell lines Huh7 or HepG2. After successful knockdown of ZIC5 validated by Western blot (Fig. 2A), the proliferation of Huh7 or HepG2 cells were detected using CCK-8. As expected, silencing ZIC5 decreased proliferation of Huh7 or HepG2 cells (Fig. 2B). Furthermore, ZIC5 knockdown and control Huh7 cells labeled with stable luciferase were subcutaneously injected into the mice, and the bioluminescence was detected using an in vivo imaging system to monitor tumor growth (Fig. 2C). As expected, ZIC5 knockdown decreased tumor incidence and the growth of Huh7 cells-derived tumor xenografts compared with the control cells (Fig. 2C). The results indicate that ZIC5 is required in growth of HCC. Matrigel invasion assays and scratch wound healing assays were tested to evaluate whether ZIC5 was responsible for the migratory and invasive capabilities of sh-ZIC5 1#-Huh7 and sh-ZIC5 1#-HepG2 cells. The Matrigel invasion assay showed that invasiveness was markedly decreased in the sh-ZIC5 1#-treated cells
Fig. 2. ZIC5 was required in proliferation, invasion, migration and tumorigenicity of HCC. A, The expression of ZIC5 in Huh7 or HepG2 cells treated with ZIC5 shRNAs or control shRNA was examined by Western blot. B, CCK-8 experiments were employed to detect the proliferation of Huh7 or HepG2 cells with ZIC5 shRNAs or control shRNA at the indicated times. C, Huh7 cells treated with ZIC5 shRNAs or control shRNA were respectively subcutaneously injected into mice (n ¼ 6/group), and the tumor incidence and growth was evaluated by bioluminescent imaging. D, The invasive properties of the cells were analyzed by an invasion assay using a Matrigel-coated Boyden chamber. E, The cell migration rates of ZIC5 shRNA-Huh7 and ZIC5 shRNA-HepG2 were tested by a wound-healing assay. Microscopic observation of wound closure was performed 0 and 24 h after scratching the cell layer. (*p < 0.05, **p < 0.01 and ***p < 0.001).
compared with the empty vector-transfected cells (sh-NC) (p < 0.05, Fig. 2D). Also, sh-ZIC5 1# decreased cell motility in HepG2 cells (Fig. 2E).
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
5
Fig. 3. ZIC5 promotes proliferation, invasion, migration and tumorigenicity of HCC cell. A, The expression of ZIC5 in Huh7 or HepG2 cells without or with ZIC5 over-expression (OE) was performed by Western blot. B, CCK-8 experiments were used to detect the proliferation of Huh7 or HepG2 cells without or with ZIC5 over-expression at the indicated times. C, Huh7 cells without or with ZIC5 over-expression were respectively subcutaneously injected into mice (n ¼ 6/group). The tumor growth was evaluated by bioluminescent imaging. D, The invasive properties of the cells were analyzed by an invasion assay using a Matrigel-coated Boyden chamber. E, The cell migration rates of ZIC5 overexpression (OE) (pcDNAZIC5) and control (Con) (pcDNA3.1 control vectors) were tested by a wound-healing assay. Microscopic observation of wound closure was performed 0 and 24 h after scratching the cell layer. (*p < 0.05, **p < 0.01 and ***p < 0.001).
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
6
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
3.3. ZIC5 promotes proliferation and tumorigenicity of HCC cells We next examined whether enforced ZIC5 facilitated the growth of HCC cells. First, ZIC5 was over-expressed (OE) in Huh7 or HepG2 cells by pcDNA-ZIC5, which was validated by Western blot (Fig. 3A). Then CCK-8 assays demonstrated that ZIC5 facilitated the proliferation of HCC cells (Fig. 3B). Second, ZIC5 over-expressed and control Huh7 cells labeled with stable luciferase were subcutaneously injected into the mice. The bioluminescence was detected using an in vivo imaging system to monitor tumor growth (Fig. 3C). As expected, ZIC5 promoted the growth of tumors derived from Huh7 cells (Fig. 3C). Compared with the empty vector-transfected cells, overexpression of ZIC5 increased cell invasiveness and migration by the matrigel invasion assay and scratch wound healing assay (p < 0.05, Fig. 3D and E). 3.4. ZIC5 promoted growth of HCC in a b-catenin dependant manner It is well-known that activated Wnt/b-catenin pathway facilitates the growth and progression of HCC. Then we determined whether ZIC5 regulatedb-catenin signaling in HCC cells. First, overexpressed ZIC5 increased the protein expression of b-catenin and CyclinD1 in Huh7 or HepG2 cells (Fig. 4A), while silencing ZIC5 decreased the b-catenin and CyclinD1 expressions in HCC cell lines
(Fig. 4B). Second, immunofluorescence assays demonstrated that over-expressed ZIC5 promptedb-catenin to enter the nucleus of HCC cells (Fig. 4C). Thus, ZIC5 activated b-catenin signaling in HCC. Furthermore, we determined whether b-catenin was required for the role of ZIC5 in HCC cell lines. CCK-8 assays presented that bcatenin knockdown alleviated the pro-tumor effects of ZIC5 in HCC cells (Fig. 4D). The results indicate that ZIC5 promotes the growth and progression of HCC via b-catenin. 4. Discussion ZIC5 has been reported to be implicated in various tumors, but there is not a literature reporting its role in HCC. Our study was the first to elucidate the expression and significance of ZIC5 in human HCC specimens, and elucidate its promoting role in growth and progression of HCC. To determine the significance of ZIC5 in patients with HCC, we first detected its expression in HCC specimens by IHC staining. Our results demonstrated that most of the HCC tissues exhibited high ZIC5 expression. Furthermore, high ZIC5 expression indicated worse overall survival and disease-free survival of HCC patients, which prompted us to examine whether ZIC5 exerted a pro-tumor function in growth of HCC. As expected, overexpressed or silencing ZIC5 respectively increased or inhibited the proliferation, migration and invasion of HCC cells in vitro and in vivo.
Fig. 4. ZIC5 promoted growth and progression of HCC in a b-catenin dependant manner. A, Western blot analysis was performed to detectb-catenin and CyclinD1 expressions in Huh7 or HepG2 cells with ZIC5 over-expression or control. B, b-catenin and CyclinD1 expressions in Huh7 or HepG2 cells with ZIC5 shRNAs or control shRNA was performed by Western blot analysis. C, Cell immunofluorescence was use to examine the location of b-catenin in the nucleus and cytoplasm of Huh7 or HepG2 cells with ZIC5 over-expression or control (scar bar ¼ 10 mm). D, CCK-8 experiments were used to assess the proliferation of Huh7 or HepG2 cells without or with ZIC5 over-expression, and with ZIC5 over-expression plus b-catenin knockdown (sh-b-catenin) at the indicated times. (*p < 0.05, **p < 0.01 and ***p < 0.001).
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009
L. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e7
Additionally, we revealed a novel mechanism underlying ZIC5 in activating b-catenin in HCC cells. Though previous study showed the b-catenin facilitated the growth and progression of in HCC [19,20], whether ZIC5 mediated b-catenin in HCC has not been elucidated. Our study indicated that b-catenin was necessary in ZIC5-regulaitng growth of HCC. Thus, ZIC5 may be served as a crucial indicator for prognosis of HCC patients and a novel target for HCC treatment. Our studies will examine how ZIC5 regulated bcatenin in HCC cells and evaluate the effect of targeting ZIC5 and related pathways on HCC treatment in vivo. CyclinD1 is cell-cycle proteins. We only tested the expression of cyclin D1 after silencing or overexpressing ZIC5. It show that CyclinD1 was regulated by ZIC5 in HCC. However, it was not known which mechanism was involved in the proliferation of HCC by ZIC5 regulation or whether it was related to the cell cycle. This is what we will continue to study later. Conflicts of interest The authors declare that they have no competing interests. Acknowledgments This work was supported by The National Natural Science Foundation of China (81400699) Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.bbrc.2018.08.009. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.08.009. References [1] L.A. Torre, F. Bray, R.L. Siegel, J. Ferlay, J. Lortet-Tieulent, A. Jemal, Global cancer statistics, Ca - Cancer J. Clin. 65 (2015) (2012) 87e108. [2] X. Sun, S.C. Wang, Y. Wei, X. Luo, Y. Jia, L. Li, P. Gopal, M. Zhu, I. Nassour, J.C. Chuang, T. Maples, C. Celen, L.H. Nguyen, L. Wu, S. Fu, W. Li, L. Hui, F. Tian, Y. Ji, S. Zhang, M. Sorouri, T.H. Hwang, L. Letzig, L. James, Z. Wang, A.C. Yopp, A.G. Singal, H. Zhu, Arid1a has context-dependent oncogenic and tumor suppressor functions in liver cancer, Canc. Cell 32 (2017) 574e589, e576. [3] C. Wang, S.Y. Fu, M.D. Wang, W.B. Yu, Q.S. Cui, H.R. Wang, H. Huang, W. Dong, W.W. Zhang, P.P. Li, C. Lin, Z.Y. Pan, Y. Yang, M.C. Wu, W.P. Zhou, Zinc finger protein X-linked promotes expansion of EpCAM(þ) cancer stem-like cells in hepatocellular carcinoma, Mol. Oncol. 11 (2017) 455e469.
7
[4] G. Ma, W. Dai, A. Sang, X. Yang, Q. Li, Roles of ZIC family genes in human gastric cancer, Int. J. Mol. Med. 38 (2016) 259e266. [5] J. Aruga, The role of Zic genes in neural development, Mol. Cell. Neurosci. 26 (2004) 205e221. [6] R. Satow, S. Inagaki, C. Kato, M. Shimozawa, K. Fukami, Identification of zinc finger protein of the cerebellum 5 as a survival factor of prostate and colorectal cancer cells, Canc. Sci. 108 (2017) 2405e2412. [7] W. Han, C. Zhang, X.J. Gao, H.B. Wang, F. Chen, F. Cao, Y.W. Hu, J. Ma, X. Gu, H.Z. Ding, Clinicopathologic and prognostic significance of the zinc finger of the cerebellum family in invasive breast cancer, J. Breast Canc. 21 (2018) 51e61. [8] R. Satow, T. Nakamura, C. Kato, M. Endo, M. Tamura, R. Batori, S. Tomura, Y. Murayama, K. Fukami, ZIC5 drives melanoma aggressiveness by PDGFDmediated activation of FAK and STAT3, Canc. Res. 77 (2017) 366e377. [9] Q. Sun, R. Shi, X. Wang, D. Li, H. Wu, B. Ren, Overexpression of ZIC5 promotes proliferation in non-small cell lung cancer, Biochem. Biophys. Res. Commun. 479 (2016) 502e509. [10] Y. Shi, J. Li, C. Chen, Y. Xia, Y. Li, P. Zhang, Y. Xu, T. Li, W. Zhou, W. Song, Ketamine modulates Zic5 expression via the Notch signaling pathway in neural crest induction, Front. Mol. Neurosci. 11 (2018) 9. [11] M.K. Nyholm, S.F. Wu, R.I. Dorsky, Y. Grinblat, The zebrafish zic2a-zic5 gene pair acts downstream of canonical Wnt signaling to control cell proliferation in the developing tectum, Development 134 (2007) 735e746. [12] H.K. Lee, E.W. Lee, J. Seo, M. Jeong, S.H. Lee, S.Y. Kim, E.H. Jho, C.H. Choi, J.Y. Chung, J. Song, Ubiquitylation and degradation of adenomatous polyposis coli by MKRN1 enhances Wnt/beta-catenin signaling, Oncogene (2018 May 1), https://doi.org/10.1038/s41388-018-0267-3 [Epub ahead of print]. [13] C. Wang, M.D. Wang, P. Cheng, H. Huang, W. Dong, W.W. Zhang, P.P. Li, C. Lin, Z.Y. Pan, M.C. Wu, W.P. Zhou, Hepatitis B virus X protein promotes the stemlike properties of OV6(þ) cancer cells in hepatocellular carcinoma, Cell Death Dis. 8 (2017), e2560. [14] X. Jiang, P.Y. Mak, H. Mu, W. Tao, D.H. Mak, S. Kornblau, Q. Zhang, P. Ruvolo, J.K. Burks, W. Zhang, T. McQueen, R. Pan, H. Zhou, M. Konopleva, J. Cortes, Q. Liu, M. Andreeff, B.Z. Carter, Disruption of Wnt/beta-catenin exerts antileukemia activity and synergizes with FLT3 inhibition in FLT3-mutant acute myeloid leukemia, Clin. Canc. Res. Off. J. Am. Assoc. Canc. Res. 24 (2018) 2417e2429. [15] Q. Chen, J. Cai, Q. Wang, Y. Wang, M. Liu, J. Yang, J. Zhou, C. Kang, M. Li, C. Jiang, Long noncoding RNA NEAT1, regulated by the EGFR pathway, contributes to glioblastoma progression through the WNT/beta-Catenin pathway by scaffolding EZH2, Clin. Canc. Res. Off. J. Am. Assoc. Canc. Res. 24 (2018) 684e695. [16] H. Clevers, Wnt/beta-catenin signaling in development and disease, Cell 127 (2006) 469e480. [17] S. Fatima, X. Shi, Z. Lin, G.Q. Chen, X.H. Pan, J.C. Wu, J.W. Ho, N.P. Lee, H. Gao, G. Zhang, A. Lu, Z.X. Bian, 5-Hydroxytryptamine promotes hepatocellular carcinoma proliferation by influencing beta-catenin, Mol Oncol 10 (2016) 195e212. [18] B. Zhou, H. Chen, D. Wei, Y. Kuang, X. Zhao, G. Li, J. Xie, P. Chen, A novel miR219-SMC4-JAK2/Stat3 regulatory pathway in human hepatocellular carcinoma, J. Exp. Clin. Canc. Res. 33 (2014) 55. [19] H. Yin, R. Que, C. Liu, W. Ji, B. Sun, X. Lin, Q. Zhang, X. Zhao, Z. Peng, X. Zhang, H. Qian, L. Chen, Y. Yao, C. Su, Survivin-targeted drug screening platform identifies a matrine derivative WM-127 as a potential therapeutics against hepatocellular carcinoma, Canc. Lett. 425 (2018) 54e64. [20] Y.F. Sun, W. Guo, Y. Xu, Y.H. Shi, Z.J. Gong, Y. Ji, M. Du, X. Zhang, B. Hu, A. Huang, G.G. Chen, P.B.S. Lai, Y. Cao, S.J. Qiu, J. Zhou, X.R. Yang, J. Fan, Circulating tumor cells from different vascular sites exhibit spatial heterogeneity in epithelial and mesenchymal composition and distinct clinical significance in hepatocellular carcinoma, Clin. Canc. Res. Off. J. Am. Assoc. Canc. Res. 24 (2018) 547e559.
Please cite this article in press as: L. Liu, et al., ZIC5 facilitates the growth of hepatocellular carcinoma through activating Wnt/b-catenin pathway, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.08.009