Elevated Expression of Zinc Finger Protein 703 Promotes Cell Proliferation and Metastasis Through PI3K/AKT/GSK- 3Β Signalling in Oral Squamous Cell Carcinoma

Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/00048536010.1159/000485360 © 2017 The Author(s).© 2017 Published The Author(s) by S. Karger AG, Basel Published online: online: November November 24,24, 2017 www.karger.com/cpbPublished by S. Karger AG, Basel 920 and Biochemistry www.karger.com/cpb Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis Accepted: October 15, 2017

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Elevated Expression of Zinc Finger Protein 703 Promotes Cell Proliferation and Metastasis through PI3K/AKT/GSK- 3β Signalling in Oral Squamous Cell Carcinoma

Hao Wanga Xubin Dengd Jinshan Zhanga Zhilin Oua Jiajie Maia Shangwei Dingc Shaofen Huob aDepartment of Radiation Oncology, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, bDepartment of otorhinolaryngology of Nanfang Hospital, Southern Medical University, Guangzhou, cDepartment of Ultrasound, Dongguan People’s Hospital Affiliated to Southern Medical University, Dongguan, Guangdong, dDepartment of Medical Oncology,Affiliated Cancer Hospital of Guangzhou Medical University, Guangzhou, PR China

Key Words Znf703 • Cell growth • Metastasis • OSCC • AKT • PI3K • GSK-3β

Abstract Background/Aims: Zinc finger protein 703 (ZNF703), initially identified as a novel oncogene in human breast cancer, is a member of the NET/NlZ family of zinc finger transcription factors. It is recognized that the overexpression of ZNF703 is associated with various types of human cancers, but the role and molecular mechanism of ZNF703 in oral squamous cell carcinoma (OSCC) are unknown. Methods: ZNF703 expression levels were examined in OSCC tissues and non-cancerous tissues by qRT-PCR and immunohistochemistry (IHC). The molecular mechanisms of ZNF703 and its effects on cell growth and metastasis were explored in vitro and in vivo using the CCK8 assay, colony formation assay, cell cycle analysis, migration and invasion assays, wound-healing assay, western blotting and xenograft experiments in nude mice. Results: In this study, ZNF703 was found to be upregulated in OSCC tissues compared to that in normal tissues at both mRNA and protein levels, and its expression level was closely correlated with the overall survival of patients with OSCC. Silencing of the ZNF703 gene in OSCC cells significantly inhibited cell growth and metastasis in vitro and in vivo. Conversely, the overexpression of ZNF703 in OSCC cells promoted cancer growth and metastasis in vitro. Mechanistically, ZNF703 activated the PI3K/AKT/GSK-3β signalling pathway and its downstream effectors, thus regulating the cell cycle and epithelial-mesenchymal transition (EMT). Furthermore, the promotive effects of ZNF703 on cellular proliferation and metastasis could be rescued by LY294002 (a PI3K-specific inhibitor) and MK2206 (an Akt-specific inhibitor). Conclusion: The results show that ZNF703 promotes cell growth and metastasis H. Wang, X. Deng and J. Zhang contributed equally to this work.

Shangwei Ding Department of Ultrasound, Dongguan People’s Hospital Affiliated to Southern Medical and Shaofen Huo University, Department of otorhinolaryngology of Nanfang Hospital, Southern Medical University, (China); E-Mail [email protected], [email protected] Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 921 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis through PI3K/Akt/GSK-3β signalling in OSCC and that it may be a promising target in the treatment of patients with OSCC. © 2017 The Author(s) Published by S. Karger AG, Basel

Introduction

Oral cancer is a malignancy of the lip or oral cavity. It is one of the 10 most common cancers in the world, and 90% of oral cancer cases are oral squamous cell carcinoma (OSCC) [1]. Surgery, radiation therapy and coadjutant therapy are common approaches used in the treatment of OSCC[2]. Despite advances in surgery and radiotherapy, which remain the standard treatment options, the prognosis of patients with OSCC is still poor, and the mortality rate has remained largely unchanged for decades [3]. Unfortunately, most of the OSCC cases are diagnosed at advanced stages (III or IV), with the 5-year overall survival rate varying between 40% and 50% and a cure rate of 30%[4-6]. Therefore, early diagnosis and treatment remain the key elements for improving the survival of patients.

Zinc finger protein 703 (ZNF703), encoded by a gene in chromosome 8p11-12, is a member of the NET family of zinc finger transcription factors [7, 8]. Members of the NET is(for overexpressed NocA, Nlz, Elbow, in several and TLP-1) types of family malignancies, have recently including been breastshown cancer to play [11], important lung cancer roles [12],in embryonic gastric cancer development [13], colorectal and tumour cancer progression [14] and [9,cholangiocarcinoma 10]. It has been shown [15], thatand ZNF703its high expression to promotes regulate thecell progressionadhesion, migration, of these and cancers. proliferation Investigations in breast have cancer confirmed [16]. that ZNF703 can complex with Groucho and repress E-cadherin, Wnt and TGF-β reporter cancer and to be an independent unfavourable prognostic factor for the survival of patients Overexpressed ZNF703 was shown to increase lung metastases in a mouse model of breast with luminal B breast cancer [11, 17]. In addition, overexpressed ZNF703 was shown to stillcontribute unclear. to tumour progression in non-small cell lung cancer by activating the Akt/mTOR signalling pathway [12]. However, the role and molecular mechanism of ZNF703 in OSCC are and Thus,that itsthis overexpression study was designed increased to explore the oncogenic the role ofpotential, ZNF703 inespecially the development proliferation, and progression of OSCC. In this study, we determined that ZNF703 was upregulated in OSCC its downstream effectors. migration and invasion, of OSCC by activating the PI3K/Akt/GSK-3β signalling pathway and

Materials and Methods

Cell culture, Sample collection, and Ethics Statement

The OSCC cell lines Tca-8113 and KB were maintained in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS) (PAA Laboratories, Inc, Pasching, Austria). The cells were incubated in a humidified chamber with 5% CO2 at 37°C. Twenty-five freshly isolated OSCC tissues, 15 freshly isolated non-cancerous tissues, 95 paraffin-embedded OSCC tissues and 24 paraffin-embedded non-cancerous specimens were obtained at the time of diagnosis before any therapy from the Third Affiliated Hospital of Guangzhou Medical University, Guangzhou. Among the 95 OSCC patients, there were 55 males and 40 consentfemales withwas obtained ages ranging from from the patients. 17 to 79 years (median, 48.18 years). All clinical protocols were approved by the Ethics Committees of the Third Affiliated Hospital of Guangzhou Medical University, and informed Immunohistochemistry

Immunohistochemistry and evaluation of ZNF703 staining (1:100, GTX107721, GeneTex) were performed in OSCC and non-cancerous oral tissues according to previously described methods [18]. Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 922 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

RNA Isolation, Reverse Transcription, and qRT-PCR

RNA was extracted from the OSCC cell lines, 22 fresh OSCC tissues and 12 fresh non-cancerous oral tissues using TRIzol (Takara, Shiga, Japan). For the evaluation of ZNF703 expression, RNA was transcribed into cDNA and amplified with the specific sense primer: 5’-GATCAGGGTCCTGAAGATGC-3’ and antisense internalprimer: 5’-CCGAGTTGAGTTTGGAGGAG-3’. control. The assays were performed in accordance with the manufacturer’s instructions (Takara). PCR amplification was repeated three times for each gene, and ARF5 was used as an Western Blot Analysis

Cells were lysed in RIPA buffer (Beyotime, China), and protein concentration was determined using the BCA kit (Beyotime, China). Total protein (25 μg) was resolved using a 10% SDS-polyacrylamide gel electrophoresis (PAGE) gel and electro-transferred onto polyvinylidene fluoride membranes (PVDF) (Millipore Corp, USA). The membranes were blocked with 5% non-fat dry milk (with BSA for phosphorylation-specific antibodies) in Tris-buffered saline (pH 7.5), followed by immunoblotting overnight at 4°C with anti-ZNF703 antibody (1:1000; GTX107721, GeneTex); anti-GSK- 3β, anti-p-GSK-3β (Ser9), anti-p-Akt (Ser473), anti-Akt, anti-p-PI3K (Tyr458) and anti-PI3K antibodies (1:1000, Cell Signalling Technology); anti-MYC, anti-CDK4, anti-CCND1, anti-E2F1, and anti-β-actin antibodies (1:500, Santa Cruz Biotechnology); and anti-MMP9, anti-Snail, anti-Vimentin, anti-N-cadherin, and anti-E-cadherin antibodies (1:1000, Cell Signalling Technology, Danvers, USA). HRP-conjugated anti- rabbit or anti-mouse IgG antibody was used as the secondary antibodies (Bio-Rad, Hercules, CA). Signals were Immunofluorescencedetected by enhanced chemiluminescence (ECL, GE Healthcare, Buckinghamshire, UK).

solutionCells was were washed cultured out, overnight the cells andwere fixed incubated with 3.5% with paraformaldehyde primary antibody atfor 4°C 2 h,for followed 30 min. byAfter incubation fixation, withthe cells secondary were permeabilized antibodies (Invitrogen) in 0.2% Triton for 1 h X-100 at room for temperature. 30 min at room Coverslips temperature. were then After mounted the blocking onto

the slidesEstablishment with mounting of stable solution OSCC containingTca-8113 and 0.2 KBmg/ml cells DAPI infected and with sealed ZNF703 with nailoverexpression polish. constructs or ZNF703-targeting shRNA constructs

- Lentiviruses carrying human ZNF703-targeting short hairpin RNA (shRNA) were generated using the- pLVTHM-GFP lentiviral RNAi expression system (sense: 5’-CGCGTCCCCGCTGTATGGACAGAGACTAGCTTCAA GAGAGCTAGTCTCTGTCCATACAGCTTTTTGGAAAT-3’ and antisense: 5’-CGATTTCCAAAAAGCTGTATGGACA GAGACTAGCTCTCTTGAAGCTAGTCTCTGTCCATACAGCGGGGA-3’). ZNF703 cDNA was cloned into an AgeI- digested pGC-FU-GFP lentiviral vector. Tca-8113 and KB cells were infected with lentiviruses carrying pGC- FU-ZNF703-GFP vector or pLVTHM-shZNF703-GFP vector as described in previous studies. Flow cytometry was usedTransient to select transfection polyclonal with cells the with Akt GFPInhibitor expression MK2206 for or subsequent the PI3K Inhibitor experiments. LY294002

The Akt inhibitor MK2206 and the PI3K inhibitor LY294002 were purchased from Sigma-Aldrich. Approximately 24 h before transfection, Tca-8113-ZNF703 and KB-ZNF703 cells were plated in 6-well plates at 35-55% confluence. The Akt inhibitor MK2206 or the PI3K inhibitor LY294002 was then transfected experiments.into cells at a working concentration of 5 µM or 50 nM, respectively, using Lipofectamine 2000 (Invitrogen Carlsbad, CA) according to the manufacturer’s protocol. After 48–72 h, cells were collected for subsequent CCK8 Assay in vitro

The CCK8 assay was used to assess cell proliferation. After transfection with pGC-FU-ZNF703 or shRNA-ZNF703, cells were seeded in 96-well plates at a density of 2, 000 cells/well and incubated for 1, 2, 3, 4, 5, 6, or 7 days. For inhibitor assays, cells were seeded in 96-well plates at a density of 2, 000 cells/well and incubated for 1, 2 or 3 days. Ten microliters of the CCK8 reagent (Beyotime, China) was added to each well and incubated for 1 h. At the end of incubation, the absorbance value (OD) of each well was measured at 450 nm. Experiments were performed three times. Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 923 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

Colony formation assay

Cells were plated in 6-well culture plates at a density of 200 cells/well and incubated for 14 days in a humidified chamber with 5% CO2 at 37°C. At the end of incubation, the cells were washed with PBS inoculated)×100%.and stained with Giemsa solution. The number of colonies containing ≥50 cells was counted under a microscope. The efficiency of colony formation was calculated using (number of colonies/number of cells Cell Cycle Analysis

5×106 Cells were seeded in 10-cm plates in RPMI-1640 containing 10% FBS and incubated for 48 h. A total of cells were harvested, washed with cold PBS, and fixed with 70% ice-cold ethanol. After 48 h, the fixed cells were washed with cold PBS and incubated with PBS containing propidium iodide (PI, 10 µg/ml) and RNase A (0.5 mg/ml) for 20 min. The DNA content of labelled cells was determined using flow cytometry (BD Biosciences).Migration and Each Invasion experiment Assays was performed three times.

cell migration assay, a total of 1×106 Cell migration and invasion assays were performed according to our previous study [18]. For the cells in medium without FBS were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning, USA). After incubation for 12 h, the cells adhering to the lower surface were fixed with methanol, stained with Giemsa solution, and counted under a microscope in five pre-determined fields. The procedure for the cell invasion assay was similar to that of the cell migration assay, except that the transwell membranes were pre-coated with 24 mg/ml Matrigel (R&D Systems,Wound-healing USA) and incubation assay for 16 h.

For the wound-healing assay, cells were seeded in 6-well plates and incubated for 24 h in a humidified chamber with 5% CO2 at 37°C. At the end of incubation, three scratch wounds were made across each well using a P-200 pipette tip. Fresh RPMI-1640 medium containing 5% FBS was added. The cells were continuously incubated and the wound closure was observed for 48 h. Photographs were captured at 0 and 48 h. In vivo tumour growth assay

The experiment was carried out according to a6 cells previously were subcutaneously described protocol injected [18]. into Briefly, the left cellsand were seeded in 10-cm plates and incubated for 48 h. At the end of incubation, cells were harvested and resuspended with serum-free medium. A total of 2×10 right dorsal regions of 12 nude mice. The mice were maintained in a barrier facility on HEPA-filtered racks and fed an autoclaved laboratory rodent diet. After 30 days, fluorescence signals from cells were evaluated with a whole-body GFP imaging system (LightTools, Encinitas, CA, USA). The mice were sacrificed. Tumour areatissues in realwere time. excised and weighed. The IPP 5.0 software (Media Cybernetics, Silver Spring, MD, USA) was used for the analysis of whole-body optical images, which were used to visualize tumour growth and tumour In vivo metastasis assay 6 cells

The experiment was carried out according to a previously described protocol [18]. Briefly, 5×10 were harvested and injected into the spleen of nude mice. After two months, all mice were sacrificed, and individual organs were excised. Metastatic tissues were analysed by H&E staining. Whole-body optical imagesStatistical were visualized Analysis to monitor primary tumour growth and formation of metastatic lesions.

used to compare the differences among groups for all in vitro experiments. The Chi-square test was used All data were analysed for statistical significance using SPSS 18.0 software. One-way ANOVA was

to analyse the relationship between ZNF703 expression levels and clinicopathological characteristics of patients. Survival analysis was performed using the Kaplan-Meier method. Two-tailed Student’s t-test was used for comparisons between two independent groups. A value of P˂0.05 was considered statistically significant. Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 924 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

Results

Upregulated expression of ZNF703 protein is unfavourable for OSCC patient prognosis

We analysed ZNF703 mRNA expression in OSCC tissues and non-cancerous tissues by real-time quantitative RT-PCR (qRT-PCR) and found that ZNF703 expression level was embeddedsignificantly OSCC increased specimens in OSCC and tissues 24 non-cancerous in comparison specimenswith the level by inimmunohistochemistry. non-cancerous tissues (Fig.1-A). We also analysed the expression levels of ZNF703 protein in 95 archived paraffin- increased in OSCC tissues in comparison with the expression in non-cancerous tissues Similar to the mRNA expression pattern, ZNF703 protein expression was significantly

(Table 1). The ZNF703 protein was localized in the cytoplasm and nucleus but mostly in the nucleus (Fig. 1-B-C). Further, we analysed the relationship between clinicopathologic characteristics and ZNF703 expression levels in individuals with OSCC (Table 2). In the 95 OSCC cases, the upregulation of ZNF703 was related to tumour size (T classification) (T1– patientT2 versus age, T3–T4), sex, smoking lymph-node status, metastasis alcohol use, (N disease classification) recurrence, (N0–N1 and versustumour N2–N3), differentiation. distant metastasis (M classification) (M0 versus M1), and clinical stage (I–II versus III–IV) but not to

In addition, we examined the association between ZNF703 protein expression levels and patient survival using Kaplan–Meier analysis with the log-rank test. The results indicated that the level of ZNF703 protein was significantly correlated with overall survival, as patients with high ZNF703 expression had significantly shorter survival than patients with negative or lowZNF703 ZNF703 promotes expression cell proliferation, (Fig. 1-D). migration and invasion in vitro and in vivo

To determine the functional significance of ZNF703 in cell proliferation, migration and invasion,Fig. 1. we first performed- loss-of-function experiments. We used lentiviruses encoding promoted Elevatedtumour ex progressionpression of and ZNF703 poor prognosis of OSCC pa- with that in non-can- tients. A. Compared cerous tissues, mRNA- creasedexpression in OSCC of ZNF703 tissues aswas shown significantly by real-time in expressionquantitative in 95 RT-PCR OSCC samples(P<0.05). by B. immuno ZNF703- a. negative expres- sionhistochemical in oral cavity staining: epi- positive nuclear and cytoplasmicthelium; b/c. expres weakly- sion in OSCC tissues; - d/e. moderately positive nuclear and cytoplasmic expression in OSCC tissues; f. strongly positive nuclear and cytoplasmic expression in OSCC tissues, mainly nuclear staining. C. KB and Tca-8113 cells were immu nostained for the localization of ZNF703 (green) with DAPI (blue) to mark the nuclei (magnification, ×1000). D. Kaplan-Meier analysis of overall survival in 95 OSCC patients according to ZNF703 protein expression. Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 925 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

Table 1. - knock down the expression of pression in OSCC shRNAs to specifically and stably Immunohistochemistry analysis of ZNF703 ex ZNF703 expression (%)

N ZNF703 in Tca-8113 and KB cells. The Group Low High P Value inexpression these cells of and ZNF703 compared protein to that was N Non-cancerous confirmed by western blot analysis 24 22(91.7%) 2(8.3%) p﹤0.0001 tissues in cells transfected with sh-con (Fig. Cancer 95 40(42.1%) 55(57.9%) formation2-A). Growth assay curves showed determined by Table 2. - the CCK8 assay and colony copathological parameters in OSCC patients (n = 95) Correlation between expression of ZNF703 protein and clini growththat suppressing and proliferation ZNF703 significantly inhibited the Variables ZNF703 (%) High n Low expression P Value inof comparison shZNF703-Tca-8113 with the expression growthand shZNF703-KB and proliferation cells Gen de r of the corresponding sh- Male 55 31(56.4%) 24(43.6%)

Female 40 24(60.0%) 16(40.0%) 0.723 transwellcon cells migration (Fig. 2-B-C). and Age（years) invasionWound-healing assays showed assay that and 37 20 (54.1%) 17(45.9%)

≥55 23(39.7% suppressed the migration ＜55 58 35 (60.3%) 0.545 silencing ZNF703 markedly ) Smoking cellsand invasionin comparison of shZNF703- with the Yes 42 25 (59.5%) 17 (40.5%) migrationTca-8113 andand shZNF703-KB invasion of No 53 30 (56.6%) 23 (43.4%) 0.775 the corresponding sh-con Differentiation we also observed that the Well-differentiated 33 23(69.7%) 10(30.3%) Moderate to poorly cells (Fig. 2-E-F). In addition, differentiated 62 32(53.3%) 28(46.7%) 0.125 cell cycle transition from Alcohol depletion of ZNF703 blocked Yes 35 19 (54.3%) 16(45.7%) cellsG1 to compared S phase in to shZNF703- that in the No 60 36(60.0%) 24(40.0%) 0.586 correspondingTca-8113 and shZNF703-KB sh-con cells Recurrence

Yes 25 15(60.0%) 10(40.0%) also altered the cytoskeletal No 70 40(57.1%) 30(42.9%) 0.804 (Fig. 2-D). ZNF703 silencing T classification organization and polarity T1+T2 65 32(49.2%) 33(50.8%) whichof were shZNF703-Tca-8113 shortened and T3+T4 30 23(76.7%) 7(23.3%) 0.012 weakenedand shZNF703-KB in comparison cells, to N classification those in the corresponding N0+N1 54 25 (46.3%) 29(53.7%)

To assess the effect of N2+N3 41 30 (73.2%) 11 (26.8%) 0.009 sh-con cells (Fig. 2-G). Distant metastasis metastasis of OSCC in vivo, M1 10 9 (90.0%) 1 (10.0%) weZNF703 studied on thetumourigenesis growth and M0 85 46 (54.1%) 39(45.9%) 0.030 in nude mice. The results Clinical stage showed that the average volume of tumours derived I～II 61 29(47.5%) 32(52.5%) III～IV 34 26 (76.5%) 8(23.5%) 0.006 from shZNF703-Tca-8113 Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 926 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

Fig. 2. Downregulated

suppressed cell migration,ZNF703 invasion expression and proliferation in vitro. - down was detected byA. western ZNF703 blotting knock in - pared to that in sh- consh-ZNF703 cells. B-C. cells In comvitro proliferative ability of OSCC cells was markedly decreased in sh-

to that in sh-con cells ZNF703 cells compared assay and colony for- mationas shown assay. by theC. Deple CCK8- - sion blocked cell cycle tion of ZNF703 expres

transition from G1 to- S phase in sh-ZNF703 expressioncells. E-F. In reduced vitro sup the migrationpression and of invasion ZNF703

of sh-ZNF703-KB and- rescencesh-ZNF703-Tca-8113 staining in cells. G. Immunofluo

sh-ZNF703-KB, sh- ZNF703-Tca-8113 and Datash-con arecells forpresented β-actin as(red) the and DAPImeans±SD (blue). of three independent experiments.

- *P<0.05, **P<0.01, and- ence***P<0.001, compared statistito the control.cally significant differ

and shZNF703-KB cells was significantly smaller than that of tumours derived from the corresponding sh-con cells (Fig. 3-H). Compared with the corresponding sh-con cells, shZNF703-Tca-8113 and shZNF703-KB cells formed fewer metastatic nodules in the livers of nude mice (Fig. 3-I). Next, we investigated whether the overexpression of ZNF703 has an opposite effect on cell proliferation, migration and invasion. We used lentiviruses carrying the ZNF703 gene to enhance the expression of ZNF703 in Tca-8113 and KB cells (Fig. 4-A). Interestingly, we observed the opposite results. The overexpression of ZNF703 enhanced the growth and Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 927 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

Fig. 3. expressionReduced decreasedZNF703 cell growth and metastasis in vivo. whole-body A. External- cence im- agesfluores of sh-

ZNF703-KB, andsh-ZNF703- sh-con cell-injectedTca-8113 mice and excised tumours obtained 30 days after implanta- - resenta- tivetion. images Rep stained sectionsof H&E- of

- primary tumours are shown. Mean weight of tumours measured 30 days after injection. B. External whole- body fluorescence images of liver were obtained 2 months after injection of cells into mouse spleen. Repre sentative images of H&E-stained sections of metastatic tumours are shown. proliferation of Tca-8113 and KB cells (Fig. 4-B-D). Furthermore, ZNF703in vitro overexpression and in vivo, andpromoted it thus theplays migration an important and role invasion in the of tumourigenesis Tca-8113 and of KB OSCC. cells (Fig. 4-E-F). Therefore, ZNF703 significantly promoted cellular growth, migration and invasion ZNF703 regulates the expression of cell cycle- and EMT-associated factors in OSCC cells

Based on the fact that ZNF703 promoted proliferation, migration and invasion in OSCC cell lines, we examined the expression of cell cycle- and EMT-associated proteins in ZNF703- downregulated cells. We found that the loss of ZNF703 blocked the expression of CDK4, CCND1, E2F1, c-Myc, MMP9, Snail, Vimentin and N-cadherin (Fig. 5-A-B). Moreover, we observedZNF703 that modulates the depletion cell of cycle ZNF703 progression increased and the expression EMT through of E-cadherin the PI3K/Akt (Fig. /GSK-3β5-A-B). signalling pathway

PI3K and AKT have been reported as upstream factors regulating cell cycle progression and EMT [19, 20]. We also examined the effect of ZNF703 on the PI3K/Akt signalling pathway and found that the loss of ZNF703 reduced the expression of phosphorylated PI3K (Tyr458), Akt (Ser473) and GSK-3 (Ser9), whereas the expression of PI3K, Akt and GSK-3β remained unchanged (Fig. 5-C). On the other hand, the specific inhibitor of PI3K LY294002 Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 928 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

Fig. 4. Upregulation of - moted cell proliferation, migrationZNF703 expressionand invasion pro

- sionin vitro. was detected A. Restoration by west of- ZNF703 protein expres cells compared to that in vector-transfectedern blotting in ZNF703cells. B-C. In vitro proliferative ability of OSCC cells was markedly increased in

compared with that in vector-transfectedZNF703-transfected cells cells as

and colony formation as- shown by the CCK8 assay expression stimulated cell say. D. Elevation of ZNF703

cycle transition from G1 to S phase in ZNF703 cells. promotedE-F. In vitro the introduction migration of ZNF703 expression

cells.and invasionData are of presented ZNF703- asKB the and means±SD ZNF703-Tca-8113 of three independent experiments.

*P<0.05, **P<0.01, and- pared***P<0.001, to the control. statistically significant difference com

was used to inhibit the expression of PI3K and found that the expression of phosphorylated PI3K (Tyr458), Akt (Ser473), and GSK-3β (Ser9) and the downstream cell cycle- and EMT- associated factors were reduced in ZNF703-upregulated cells (Fig. 6-A). Moreover, we used Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 929 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

Fig. 5.

ZNF703 regulated the PI3K/AKT/ GSK-3β signalling pathway and the downstream cell cycle- and EMT-associated factors. A. Silencing ZNF703 expression reduced the expression of oncogenic cell cycle regulators including c-Myc, CDK4, CCND1 and E2F1. B. Downregulating ZNF703 expression reduced the expression of EMT-associated factors including Vimentin, Snail, N-cadherin and MMP9 but induced the expression of E-cadherin. C. Knocking down ZNF703 expression decreased the expression of p-PI3K (Tyr458), p-Akt (Ser473), and p-GSK-3β (Ser9), whereas the levels of total PI3K, Akt and GSK-3β remained unchanged. β-actin served as the internal control. the specific inhibitor of Akt MK2206 to suppress the expression of Akt and found that the expression of phosphorylated Akt (Ser473) and GSK-3β (Ser9) and the downstream cell cycle- and EMT-associated factors were reduced in ZNF703-upregulated cells (Fig. 6-A). In addition, we observed that treatment with LY294002 and MK2206 suppressed the proliferative, migratory and invasive potentials of ZNF703-overexpressing cells (Fig. 6-B-C). signallingTaken pathway together, in we OSCC provide cells. compelling evidence that attenuated ZNF703 expression suppresses cell proliferation, migration and invasion by inactivating the PI3K/Akt/GSK-3β

Discussion expression of multiple genes involved in transcriptional regulation, stem cell modulation, ZNF703, a nuclear protein, is an oncogenic transcription factor that regulates the and proliferation and invasion in cancer cells [10, 21, 22]. The overexpression of ZNF703 is thought to play an important role in various human cancers [12, 13, 15, 23]; however, the role and molecular mechanism if ZNF703 are not well understood in OSCC. Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 930 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

Fig. 6. Inhibition pathwayof the PI3K/AKT/ reduced GSK-3β signalling- ed proliferation andZNF703-mediat metastasis. -

A. The PI3K inhibitor LY294002 suppressedand the Akt the in expressionhibitor MK2206 of p-

PI3K (Tyr458), (Ser9)p-Akt and (Ser473), the downstreamand p-GSK-3β cell associated factors cycle- and EMT- including CCND1, cadherinE2F1, and expres Snail- but elevated E- upregulated cells. sion in ZNF703- as the internal con- β-actin was used overexpressing cellstrol. B-C.treated ZNF703- with

- LY294002 and cellMK2206 viability were subas- say,jected and to thetranswell CCK8 migration and - sion assays. Data areMatrigel presented inva as the means±SD of three independent experiments. statistically*P<0.05, **P<0.01, sig- and ***P<0.001, compared to the control.nificant difference Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 931 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

In this study, we assessed the expression level of ZNF703 in OSCC tissues and non- cancerous tissues by immunohistochemistry and qRT-PCR and found for the first time that ZNF703 expression was significantly increased in OSCC tissues compared with that in non- cancerous tissues at both mRNA and protein levels. Importantly, IHC staining showed that in OSCC cells, the ZNF703 protein is mainly expressed in the nucleus and partly in the cytoplasm and membrane. Furthermore, we analysed the correlation between ZNF703 expression and the clinicopathological characteristics of 95 OSCC patients and found that elevated ZNF703 expression was positively associated with T classification, N classification, distant metastasis and clinical stages of OSCC in patients. Moreover, we also observed that the high expression of ZNF703 in OSCC was negatively correlated with overall patient survival. Patients with higher expression of the ZNF703 protein had a shorter overall survival. All these results indicate that overexpression of ZNF703 may be an independent unfavourable prognostic factor for OSCC patients, which is consistent with the findings of previous studies examining the role of ZNF703 in some tumours [11, 14]. Subsequently, we demonstrated that ZNF703 plays an oncogenic role in OSCC, especially andin tumour invasion proliferation, in vitro and migrationin vivo and invasion. Notably, the stable silencing of ZNF703 expression in Tca-8113 and KB cells significantly inhibited cellular proliferation, migrationin vitro. . In contrast, the stable overexpression of ZNF703 in Tca- 8113 and KB cells markedly enhanced cellular proliferation, migration and invasion Altogether, these results suggested that ZNF703 promoted cell growth and metastasis in squamousOSCC. Although cell carcinoma ZNF703 has remain been largelyshown unknown.to play crucial It is roleswell known in OSCC, that the a precisehigh proliferative molecular activitymechanisms of tumour through cells which is associated ZNF703 with affects increased proliferation, cell cycle migration progression and [24-26]. invasion Here, in oral we found that downregulating ZNF703 expression blocked cell growth by suppressing cell cycle progression and repressing the expression of cell cycle G1/S checkpoint factors, including CDK4, CCND1, c-Myc and E2F1. Epithelial-mesenchymal transition (EMT) is significantly associated with the aggressiveness and metastatic potential of cancers [27-29]. The hallmark of EMT is the loss of the epithelial marker E-cadherin [30], gain of the mesenchymal marker N-cadherin [31], and the upregulation of transcriptional repressors, such as Zeb1, Twist, Snail and Slug [32, 33]. In this study, ZNF703 overexpression promoted EMT by upregulating the expression of EMT-associated markers N-cadherin, vimentin, snail and Slug and by downregulating the expression of E-cadherin. Consistent with previous studies in which ZNF703 overexpression promoted tumour metastasis in breast cancer by repressing the expression of E-cadherin [16], we reached similar conclusions in OSCC. In addition, ZNF703 downregulation reduced the expression of MMP9, a member of the matrix metalloproteinase (MMP) family that accelerates the aggressiveness of many tumours [34, 35]. The PI3K/AKT/GSK-3β pathway is a classical signalling pathway in tumourigenesis [36, 37], and its activation upregulates the expression of Snail and Slug, thereby promoting EMT[20, 38, 39] and inducing the transition from G1 to S phase [40, 41]. In this study , we found that the loss of ZNF703 expression decreased the expression of p-PI3K, p-Akt, and p-GSK-3β. These results are similar to the findings of the study by Baykara et al. in which ZNF703 was shown to contribute to tumour development in NSCLC by activating the Akt/ mTOR pathway [12]. To specifically determine the effects of PI3K and Akt on the ZNF703- mediated promotion of cell proliferation and metastasis, we father employed the Akt inhibitor MK2206 and the PI3K inhibitor LY294002 to inhibit the expression of Akt and PI3K, respectively, in ZNF703-upregulated cells. Finally, we found that the PI3K inhibitor LY294002 inhibited the expression of phosphorylated PI3K (Tyr458), Akt (Ser473), and GSK-3β (Ser9) and the downstream cell cycle- and EMT-associated factors and that the Akt- specific inhibitor MK2206 suppressed the expression of phosphorylated Akt (Ser473) and GSK-3β (Ser9) and the downstream cell cycle- and EMT-associated factors. Cellular Physiology Cell Physiol Biochem 2017;44:920-934 DOI: 10.1159/000485360 © 2017 The Author(s). Published by S. Karger AG, Basel and Biochemistry Published online: November 24, 2017 www.karger.com/cpb 932 Wang et al.: ZNF703 Promotes OSCC Cell Proliferation and Metastasis

Conclusion

This study shows that the expression of ZNF703 is upregulated in OSCC tissues and that the overexpression of ZNF703 is significantly associated with poor prognosis in OSCC factorspatients. in Furthermore,OSCC. ZNF703 promoted cell proliferation and metastasis by activating the PI3K/AKT/GSK-3β signalling pathway and downstream cell cycle- and EMT-associated

Acknowledgements

This study was supported by Natural Science Foundation of Guangdong Province,China( NO.2015A030310111);Guangzhou Medical University Doctor Startup Project (NO.2014C38);General project of Science and Technology from Guangzhou City (NO.201707010234) and the National Natural Science Foundation of China (NO.81502576). Disclosure Statement

The authors declare no Disclosure Statement.

References

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