ZNF677 Suppresses Akt Phosphorylation and Tumorigenesis in Thyroid Cancer Yujun Li1, Qi Yang1, Haixia Guan2, Bingyin Shi1,3, Meiju Ji4, and Peng Hou1,3

Published OnlineFirst July 11, 2018; DOI: 10.1158/0008-5472.CAN-18-0003

Cancer Molecular Cell Biology Research

Abstract

The zinc finger protein 677 (ZNF677) belongs to the zinc apoptosis. Conversely, knockdown of ZNF677 promoted thy- finger protein family, which possesses transcription factor roid cancer cell proliferation and colony formation. ZNF677 activity by binding sequence-specific DNA. Previous studies exerted its tumor suppressor functions in thyroid cancer cells have reported its downregulated by promoter methylation in through transcriptional repression of two targets CDKN3 and non–small cell lung cancer. However, its biological role and HSPB1 (or HSP27), thereby inhibiting phosphorylation and exact mechanism in human cancers, including thyroid cancer, activation of Akt via distinct mechanisms. Taken together, our remain unknown. In this study, we demonstrate that ZNF677 data show that ZNF677 functions as a tumor suppressor and is frequently downregulated by promoter methylation in is frequently silenced via promoter methylation in thyroid primary papillary thyroid cancers (PTC) and show that cancer. decreased expression of ZNF677 is significantly associated with poor patient survival. Ectopic expression of ZNF677 in Significance: These findings report a tumor suppressive thyroid cancer cells dramatically inhibited cell proliferation, role of the zinc-finger protein ZNF677 in primary papillary colony formation, migration, invasion, and tumorigenic thyroid cancer through inhibition of Akt phosphorylation. potential in nude mice and induced cell-cycle arrest and Cancer Res; 78(18); 5216–28. 2018 AACR.

Introduction compelling need for developing additional therapeutic targets and treatment options. The incidence of thyroid cancer has increased rapidly in recent An increasing body of evidence suggests that epigenetic years, especially in women, and has become one of the highest in changes such as DNA methylation, remodeling, and posttrans- the world including China (1, 2). Generally, the majority of the lational modification of chromatin play critical roles in thyroid patients with well-differentiated thyroid cancer (WDTC) have an tumorigenesis, as a result of their effects on tumor cell differ- excellent prognosis and can be efficiently cured by conventional entiation, proliferation, and survival (4–6). Of these epigenetic therapies (surgery with or without radioiodinated therapy and alterations, promoter methylation is widely investigated in suppression therapy with thyroid hormone); however, some human cancers and considered as one of the major mechanisms patients can develop advanced disease such as metastatic differ- to inactivate tumor suppressor genes (7, 8). So far, many genes entiated thyroid cancer (DTC), poorly differentiated thyroid have been identified to be transcriptionally inactivated by cancer (PDTC), and anaplastic thyroid cancer (ATC) that fail to promoter methylation in different types of cancer including respond to conventional therapies, resulting in morbidity and thyroid cancer (9). Zinc finger proteins are the largest tran- mortality (3). The mechanisms underlying dedifferentiation of scription factor family in human genome, which have a wide thyroid cancer are incompletely understood. Thus, there is a variety of functions in human diseases including cancers through transcriptionally activating or repressing their targets (10). The majority of zinc finger proteins contain Kruppel€ associated box (KRAB) domains, which has been demonstrated 1Department of Endocrinology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, P.R. China. 2Department of Endocrinology and Metabolism, The to repress the transcription of their downstream targets (11). As € fi First Affiliated Hospital of China Medical University, Shenyang, P.R. China. 3Key amemberoftheKruppel C2H2-type zinc nger protein family, Laboratory for Tumor Precision Medicine of Shaanxi Province, The First Affiliated the zinc finger protein 677 (ZNF677)istheleaststudied,andis Hospital of Xi'an Jiaotong University, Xi'an, P.R. China. 4Center for Translational onlyfoundtobemethylatedinnon–smallcelllungcancer Medicine, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, P.R. (NSCLC;ref.12).However,itsroleinhumancancers,including China. thyroid cancer, remains totally unclear. Note: Supplementary data for this article are available at Cancer Research In this study, we observe that ZNF677 is frequently down- Online (http://cancerres.aacrjournals.org/). regulated by promoter methylation in papillary thyroid cancers Corresponding Authors: Peng Hou, Department of Endocrinology, The First (PTC) and find a significant association of decreased expression Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, P.R. China. Phone: of ZNF677 with poor patient survival. Functional studies show 8629-8532-4749; Fax: 8629-8532-4039; E-mail: [email protected]; and Meiju Ji, that ZNF677 plays a tumor suppressor role in thyroid cancer cells. [email protected]. Mechanistically, ZNF677 inhibits thyroid tumorigenesis through doi: 10.1158/0008-5472.CAN-18-0003 transcriptionally repressing its downstream targets CDKN3 and 2018 American Association for Cancer Research. HSPB1, and subsequently inhibiting Akt phosphorylation.

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Materials and Methods Expression plasmids, short interfering RNAs, and lentivirus transfection Clinical samples ZNF677 expression plasmid pcDNA3.1()A-ZNF677 and A total of 178 paraffin-embedded PTCs and 23 normal empty vector pcDNA3.1()AwithaMyc-Histag(Invitrogen) thyroid tissues as control subjects were randomly obtained were obtained from Yingrun Biotechnology, Co., Ltd. The from the First Affiliated Hospital of Xi'an Jiaotong University. primers used for pcDNA3.1-ZNF677 (-CDKN3 or -HSPB1) Clinicopathologic characteristics of the tumors were shown in plasmid construction were shown in Supplementary Table Supplementary Table S1. Moreover, 17 pairs of frozen surgical S5. Cells were transfected with the indicated constructs at PTC and matched noncancerous tissues were also obtained 70% confluence using X-tremeGENE HP DNA Transfection from the First Affiliated Hospital of Xi'an Jiaotong University. Reagent (Invitrogen). These patients did not receive any therapeutic intervention, and Oligonucleotides of siRNA targeting ZNF677, HSPB1, and signed an informed consent before surgery. All of the tissues CDKN3, and control siRNA were obtained from Gene Pharma were histologically examined by two senior pathologists at and Ribobio, respectively. The sequences were presented in Sup- Department of Pathology of the Hospital based on World plementary Table S6. Cells were transfected at 50% confluence Health Organization (WHO) criteria. The study was conducted using Lipofectamine 2000 (Invitrogen) with a final siRNA con- in accordance with the Declaration of Helsinki, and the pro- centration of 50 nmol/L. tocol was approved by the Institutional Review Board and Lentivirus encoding ZNF677 (PHBLV-GFP-Puro-ZNF677), Human Ethics Committee of the First Affiliated Hospital of control lentivirus (PHBLV-GFP-Puro), ZNF677-shRNA (sh- Xi'an Jiaotong University. ZNF677), and control shRNA (sh-NC) were obtained from Han- Bio Biotechnology Co., Ltd. shRNA sequences were also shown in RNA extraction and quantitative RT-PCR Supplementary Table S6. Cells were transfected at 50% confluence RNA isolation, cDNA preparation and qRT-PCR were per- with a final lentivirus multiplicity of infection (MOI) of 20–100 formed as described previously (13). The mRNA expression of for PHBLV and MOI of 20 for shRNAs. the indicated genes was normalized to 18S rRNA cDNA. Each sample was run in triplicate. The primer sequences were presented Cell proliferation, colony formation, cell cycle, apoptosis, in Supplementary Table S2. migration, and invasion assays The procedures for cell proliferation, colony formation, cell- cycle distributions, apoptosis, migration, and invasion assays DNA extraction, methylation-specific PCR, and pyrosequencing were similarly performed as described previously (17). Genomic DNA from cell lines was isolated using a standard phenol–chloroform protocol. Genomic DNA was isolated from Western blot analysis paraffin-embedded tissues as described previously (14). The The detailed protocol was performed as described previously protocols of sodium bisulfite treatment, MSP, and pyrosequen- (17). The antibody information was presented in Supplementary cing were performed as described previously (13, 15). The primer Table S7. sequences for MSP and pyrosequencing were presented in Sup- plementary Tables S2 and S3. Dual-luciferase reporter assay The promoter regions of CDKN3 and HSPB1 genes were Cell lines and drug treatments inserted into predigested pGL3-Basic luciferase vector (Promega) Human thyroid cancer cell lines BCPAP, FTC133, 8505C, to produce the luciferase reporter plasmids pGL3-CDKN3-Luc K1, and TPC1 were provided by Dr. Haixia Guan (The First and pGL3-HSPB1-Luc. The constructs were verified by Sanger Affiliated Hospital of China Medical University, Shenyang, sequencing. The primers for plasmid constructs were presented China). C643 was obtained from Dr. Lei Ye (Ruijin Hospital, in Supplementary Table S8. C643 and 293T cells were transfected Shanghai, China). All cell lines used in this study were authen- with pcDNA3.1(-)A-ZNF677 or empty vector in 24-well plates, ticated by short tandem repeat analysis in Genesky Co. Ltd, and were cotransfected with pGL3-CDKN3-Luc or pGL3-HSPB1- and the result was consistent with a previous study (16) and Luc, and pRL-TK plasmids (Promega) using Lipofectamine 2000 database (COSMIC: https://cancer.sanger.ac.uk/cell_lines; Sup- (Invitrogen). Cells were collected 48 hours posttransfection, and plementary Table S4). Meanwhile, we demonstrated that these luciferase activities were analyzed on EnSpire Multimode Plate cells were not contaminated by Mycoplasma using One- Reader (PerkinElmer) using the dual-luciferase reporter assay Step Quickcolor Mycoplasma Detection Kit (Shanghai Yise system (Promega). Data were expressed as relative luciferase Medical Technology Co., Ltd.). These cells were all routinely activity (Firefly luciferase activity/Renilla luciferase activity). Each cultured at 37C in RPMI1640 or DMEM/Ham F-12 medium experiment was performed in triplicate. with 10% FBS. To explore epigenetic mechanism of ZNF677 inactivation, cells were either treated with 5 mmol/L DNA Chromatin immunoprecipitation assay methyltransferase (DNMT) inhibitor 5-aza-20-deoxycytidine The chromatin immunoprecipitation (ChIP) assay was used to (5-Aza-dC; Sigma-Aldrich) for 5 days or 7.5 mmol/L histone evaluate transcription factor ZNF677 binding to its target DNA deacetylase (HDAC) inhibitor suberoylanilide hydroxamic using the Pierce Magnetic ChIP Kit (Pierce Biotechnology). The acid (SAHA; Cayman Chemical) for 3 days. The medium and protocol was performed as described previously (18). The DNA agent were replenished every 24 hours. In some experiments, fragments were used as templates for ChIP-qPCR analysis using cells were treated with 20 mmol/L proteasome inhibitor the primers presented in Supplementary Table S9, and data were MG132 (Selleck Chemicals) for 4 hours. The same volume of normalized by respective 5% input. Each experiment was per- the vehicle was used as the control. formed in triplicate.

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Coimmunoprecipitation control subjects. MSP results of two representative PTC samples Cells were transfected with the indicated constructs, or trans- were shown in Fig. 1E (top). Given that ZNF677 methylation was fected with the indicated siRNAs. After 48 hours, protein extracts more frequent in PTCs than control subjects, we further investi- were generated by lysing cells with RIPA buffer for 1 hour at 4C. gated the association of ZNF677 methylation with clinicopatho- Coimmunoprecipitation (co-IP) assay was then performed as logic characteristics of patients with PTC. The results showed that described previously (19). ZNF677 methylation was significantly positively associated with tumor stages (P ¼ 0.019; Supplementary Table S1). Animal studies Next, we similarly detected ZNF677 methylation in a panel of Three- to 4-week-old female athymic mice were purchased from thyroid cancer cell lines using MSP assay. As expected, complete or SLAC laboratory Animal Co., Ltd. The mice were randomly partial ZNF677 methylation was found in all of six thyroid cancer divided into two groups (6 mice/group). Tumor xenografts were cell lines (Fig. 1E, bottom). Accordingly, we found that ZNF677 established by subcutaneous inoculation of 6 106 K1 cells stably was silenced or downregulated in these cells (Supplementary Fig. expressing ZNF677 or control cells as well as K1 cells stably S1A, top). To further determine whether ZNF677 is inactivated by transfected by sh-ZNF677 or sh-NC into the right armpit region epigenetic mechanisms, these cells were treated with the DNMT of nude mice. From day 3 postinjection, tumor size was measured inhibitor 5-Aza-dC or the HDAC inhibitor SAHA, respectively. As every 2 days. Tumor volumes were calculated by the formula shown in Supplementary Fig. S1A (middle and bottom panels) (length width2 0.5). The mice were sacrificed after 15 days, and S1B, 5-Aza-dC treatment dramatically restored ZNF677 and tumors were harvested and weighted. Next, tumors obtained expression in all cell lines, and SAHA treatment also induced from representative animals were embedded in paraffin and ZNF677 reexpression in some of these cell lines. In addition, sectioned at 5 mm until use. All experimental procedures involving pyrosequencing analysis showed that 5-Aza-dC treatment animals were conducted in accordance with Institution Guide- decreased methylation levels of all 11 CpG sites within ZNF677 lines and were approved by the Laboratory Animal Center of Xi'an promoter in the indicated cell lines relative to the controls (Fig. Jiaotong University (Xi'an, P.R. China). 1F), further confirming the role of promoter methylation in ZNF677 inactivation. By analyzing the data from qRT-PCR and fi IHC pyrosequencing assays, we found that there was a signi cant IHC assay was used to evaluate the expression levels of ZNF677, negative correlation between methylation levels and mRNA ZNF677 r ¼ CDKN3, HSPB1, p-Akt Ser473, p53, and Ki67 in the xenograft expression of in a panel of thyroid cancer cells ( P ¼ tumors. The protocol was performed as described previously (18). 0.58, 0.049; Supplementary Fig. S1C). Collectively, our findings indicate that promoter methylation directly mediates transcriptional silencing of ZNF677 in thyroid cancer cells. Statistical analysis Similarly, by analyzing the TCGA and MethHC datasets, we also Gene expression in tumor tissues and control subjects were found that ZNF677 was frequently methylated and downregu- compared by the unpaired t test, and paired samples were com- lated in other cancers including lung squamous cell carcinoma pared by the paired t test. The correlation between promoter (LUSC), lung adenocarcinoma (LUAD), and breast cancer (BRCA; methylation of ZNF677 and clinicopathologic characteristics of Supplementary Fig. S2A and S2B), and a significant correlation patients with PTC was analyzed by c2 test. Survival curves were between decreased expression of ZNF677 and poor patient sur- constructed according to the Kaplan–Meier method, and statis- vival in LUAD (HR ¼ 1.391; P ¼ 0.04) and BRCA (HR ¼ 1.613, P ¼ tical analysis was performed using the log-rank test. All statistical 0.03; Supplementary Fig. S2C). These observations suggest that analyses were performed using the SPSS statistical package (11.5). ZNF677 may be a potential tumor suppressor in human cancers P values <0.05 were considered significant. including thyroid cancer, and its inactivation by promoter methylation may be a common molecular event in these cancers. Results ZNF677 is frequently downregulated by promoter methylation ZNF677 inhibits thyroid cancer cell growth in PTCs To elucidate the role of ZNF677 in thyroid tumorigenesis, a We first examined mRNA and protein expression of ZNF677 in series of in vitro experiments were performed with gain-of-func- 17 PTCs and their matched noncancerous tissues (control sub- tion and loss-of-function of ZNF677 in thyroid cancer cells. Our jects) by qRT-PCR and Western blot assays. As shown in Fig. 1A results showed that ZNF677 reexpression in K1, BCPAP, and TPC1 and B, ZNF677 was significantly downregulated in PTCs com- cells was confirmed by Western blot analysis (Fig. 2A), and ectopic pared with control subjects. This was further supported by The expression of ZNF677 significantly inhibited cell proliferation Cancer Genome Atlas (TCGA) RNA-Seq database that ZNF677 compared with the control (Fig. 2B). The inhibitory effect of expression in PTCs was significantly lower than that in normal ZNF677 on cell growth was further confirmed by colony forma- controls or matched normal thyroid tissues (Fig. 1C). Next, we tion assay. As shown in Fig. 2C, the colonies formed in ZNF677- attempted to evaluate the association of ZNF677 expression with transfected cells were fewer than those formed in vector-trans- patient survival using TCGA dataset. The results showed that fected cells. We also evaluated the effect of ZNF677 reexpression decreased expression of ZNF677 was significantly associated with on thyroid cancer cell-cycle contributions and apoptosis. As poor patient survival (HR ¼ 2.680, P ¼ 0.044; Fig. 1D). Evidently, shown in Fig. 2D, compared with vector-transfected cells, cell promoter methylation is a major inactivation mechanism of cycle was arrested at the G0–G1 phase in ZNF677-transfected cells. tumor suppressor genes in tumorigenesis (7, 8). We next assessed In addition, we found that ZNF677 transfection showed an ZNF677 methylation in PTCs by using MSP assay. Our data increase in both early and late apoptosis in comparison with showed that ZNF677 methylation was found in 35 of 178 vector transfection (Fig. 2E). Conversely, ZNF677 depletion in (19.7%) PTCs, whereas it was only found in 1 of 23 (4.3%) FTC133 and 8505C cells using two different ZNF677 siRNAs

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Figure 1. Frequent downregulation of ZNF677 by promoter methylation in PTCs. A, qRT-PCR assay was performed to assess mRNA expression of ZNF677 in primary PTCs and their matched noncancerous thyroid tissues (MN; n ¼ 17). ZNF677 expression was normalized to 18S rRNA levels. The data were compared using two-tailed paired t test. B, ZNF677 expression was determined by Western blot analysis in 8 pairs of primary PTCs and their matched noncancerous thyroid tissues (MN; left). GAPDH was used as loading control. Shown in the right panels is the quantitative illustration of the levels of ZNF677 proteins using densitometry to measure the density of the corresponding bands on the Western blot analysis shown on the left. C, Low expression of ZNF677 in PTCs was relative to normal thyroid tissues (NT) and matched normal thyroid tissues (MN) in TCGA dataset. Horizontal lines indicate the median. D, A significant association of decreased expression of ZNF677 with poor survival of patients with PTC (n ¼ 511) in TCGA dataset. E, Promoter methylation of ZNF677 in PTCs (top) and thyroid cancer cell lines (bottom) was determined by the MSP assay. In vitro methylated DNA was used as positive control for methylated gene (Pos); bisulfite-modified normal leukocyte DNA

was used as positive control for unmethylated gene (Neg); H2O was used as blank control to confirm the specificity of MSP. Mk, DNA marker; M, methylated gene; U, unmethylated gene. PTC-1 and -2 present two representative PTCs with different methylation status of ZNF677. F, Detailed pyrosequencing assay confirmed pharmacologic demethylation of ZNF677 promoter by 5-Aza-dC in BCPAP, FTC133, and K1 cells. Ctr, control; 5-Aza, 5-Aza-dC.

(si-ZNF677-275 and -587) significantly promoted cell proliferation These findings suggest that there is a close correlation between and colony-forming ability relative to control siRNA (si-NC; aberrant expression of ZNF677 and metastatic phenotypes of Fig. 2F–H). Altogether, these findings support tumor suppressor thyroid cancer cells. role of ZNF677 in thyroid cancer cells. ZNF677 inhibits Akt phosphorylation through ZNF677 inhibits thyroid cancer cell migration and invasion transcriptionally repressing CDKN3 and HSPB1 in thyroid We next assessed the effect of ZNF677 on thyroid cancer cell cancer cells migration and invasion. As shown in Supplementary Fig. S3A, To explore molecular mechanisms underlying tumor-suppres- there were a significantly lower number of migrated cells in the sive effects of ZNF677 in thyroid cancer, we first tested the effect of ZNF677-transfected cells than that in vector-transfected cells. In ZNF677 on the activity of PI3K/Akt and MAPK/Erk pathways, addition, by in vitro invasion assay, we found that that ectopic which play a crucial role in thyroid tumorigenesis (20). The results expression of ZNF677 in thyroid cancer cells significantly showed that ectopic expression or knockdown of ZNF677 almost decreased the ability of cells to pass through the Matrigel-coated did not affect Erk phosphorylation (Supplementary Fig. S4). membrane compared with the control (Supplementary Fig. S3B). However, ectopic expression of ZNF677 in K1, TPC1, and BCPAP

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Figure 2. ZNF677 inhibits thyroid cancer cell growth in vitro. A, Ectopic expression of ZNF677 was confirmed by Western blot analysis in K1, BCPAP, and TPC1 cells. GAPDH was used as loading control. B, ZNF677 transfection significantly inhibited thyroid cancer cell proliferation relative to vector transfection. C, Ectopic expression of ZNF677 significantly inhibited colony formation in the indicated cells. Left, the representative images of colony formation in the cells transfected with the indicated constructs. Quantitative analysis of colony numbers is shown on the right. D and E, Cells were first transiently transfected with the indicated constructs. Cell-cycle distribution and apoptosis were then analyzed by flow cytometry. The data were presented as mean SD of values from three independent experiments. F, ZNF677 knockdown by using two different siRNAs (si-ZNF677–275 and -587) in FTC133 and 8505C cells was evidenced by Western blot analysis. GAPDH was used as loading control. G and H, ZNF677 knockdown significantly promoted thyroid cancer cell proliferation and colony formation. The data are presented as mean SD. Statistically significant differences are indicated: , P <0.05; , P <0.01; , P <0.001.

cells significantly inhibited Akt phosphorylation at Akt Ser473 but observed that ZNF677 had little impact on p53 expression, but not at Thr308 (Fig. 3A; Supplementary Fig. S5A). On the contrary, still remained upregulated p21 expression in BCPAP and 8505C ZNF677 knockdown promoted phosphorylation of Akt Ser473, cells expressing mutant p53 (Fig. 3A and B; Supplementary Fig. but not Akt Thr308 (Fig. 3B; Supplementary Fig. S5B). It is clear that S5A and S5B), suggesting that ZNF677 upregulates p21 expres- p53 stability is regulated by MDM2-mediated ubiquitination and sion and induces G0–G1 phase arrest through the p53-indepen- the ubiquitin–proteasome system via Akt cascade (21, 22). dent pathway in mutated p53 cells. Indeed, our data showed that ectopic expression of ZNF677 A previous study has identified a number of genes negatively upregulated the expression of p53 and its downstream target regulated by ZNF677 in NSCLC cells (12). Of them, CDKN3 and p21 in wild-type p53 thyroid cancer cell lines K1 and TPC1 (Fig. HSPB1 have attracted our attention because of their role in the 3A; Supplementary Fig. S5A). Conversely, ZNF677 depletion stimulation of Akt phosphorylation (23–25). We thus speculate downregulated the expression of p53 and p21 in these two cell that ZNF677 inhibits Akt phosphorylation through transcription- lines (Fig. 3B; Supplementary Fig. S5B). In addition, we also ally repressing these two genes. To do this, we first determined

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Figure 3. ZNF677 inhibits phosphorylation of Akt Ser473 by negatively regulating CDKN3 and HSPB1. A and B, Cells transfected with the indicated constructs and siRNAs were lysed, and the lysates were subjected to Western blot analysis. The antibodies against ZNF677, phospho-Akt Ser473 (p-Akt Ser473), phospho-Akt Thr308 (p-Akt Thr308), total Akt (t-Akt), p53 and p21 were used to determine the effect of ectopic expression of ZNF677 on Akt phosphorylation and p53/p21 signaling. GAPDH was used as loading control. C and D, Cells were transfected with the indicated constructs and siRNAs, qRT-PCR, and Western blot assays were then performed to determine the effect of ZNF677 on the expression of CDKN3 and HSPB1 at both mRNA and protein levels. mRNA expression of ZNF677 was normalized to 18S rRNA levels. GAPDH was used as loading control for Western blot analysis. E and F, Cells were transfected with the indicated constructs. Western blot analysis was performed to evaluate the effect of CDKN3 and HSPB1 on Akt phosphorylation and p53 signaling. GAPDH was used as loading control. si-NC, control siRNA. G, Cells stably expressing ZNF677 and control cells were transfected with the indicated constructs (pcDNA3.1-CDKN3 and -HSPB1). Cell proliferation was then evaluated by MTT assay. The data are presented as mean SD. H, The effect of ectopic expression of CDKN3 and HSPB1 in cells stably expressing ZNF677 on Akt phosphorylation and p53/p21 signaling. GAPDH was used as loading control. Statistically signiﬁcant differences are indicated: , P <0.05; , P <0.01; , P <0.001.

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transcriptional repression of CDKN3 and HSPB1 by ZNF677 in cells was signiﬁcantly attenuated upon CDKN3 or HSPB1 reex- thyroid cancer cells. As shown in Fig. 3C and D and Supplemen- pression (Fig. 3G). In addition, we also found that ectopic tary Fig. S5C, ectopic expression of ZNF677 dramatically inhib- expression of CDKN3 or HSPB1 in K1 and TPC1 cells reversed ited the expression of CDKN3 and HSPB1 at both mRNA and inhibitory effect of ZNF677 on Akt phosphorylation, and its protein levels, while ZNF677 knockdown signiﬁcantly upregu- promoting effect on the expression of p53 and p21 (Fig. 3H; lated the expression of these two genes. Next, we tested the effect Supplementary Fig. S5F). Conversely, proliferation-promoting of CDKN3 and HSPB1 on Akt phosphorylation and the expres- effect of ZNF677 knockdown on thyroid cancer cells was signif- sion of p53 and p21 in K1 and TPC1 cells. As expected, our data icantly attenuated upon CDKN3 or HSPB1 knockdown (Supple- showed that ectopic expression of CDKN3 and HSPB1 in these mentary Fig. S6A). In addition, we also found that knocking down cells induced Akt phosphorylation, and downregulated the CDKN3 or HSPB1 in K1 and TPC1 cells partially reversed pro- expression of p53 and p21 relative to the control (Fig. 3E and moting effect of ZNF677 downregulation on Akt phosphoryla- F; Supplementary Fig. S5D and S5E). To further determine the role tion, and its inhibitory effect on the expression of p53 and p21 of CDKN3 or HSPB1 in tumor-suppressive effect of ZNF677 on (Supplementary Fig. S6B and S6C). Similar effects of ZNF677 on thyroid cancer cells, we ectopically expressed CDKN3 or HSPB1 in Akt phosphorylation and the expression of p53 and p21 were also K1 and TPC1 cells stably expressing ZNF677. The results showed found in lung cancer cell line A549, gastric cancer cell line AGS, that proliferation-inhibiting effect of ZNF677 on thyroid cancer and breast cancer cell line MCF7 (Supplementary Fig. S7).

Figure 4. Identification of CDKN3 and HSPB1 as downstream targets of ZNF677. A, ZNF677 expression was significantly negatively correlated with the expression of CDKN3 (r ¼0.27; P <0.0001) and HSPB1 (r ¼0.4; P <0.0001) in PTCs (data from TCGA database). B, C643 cells stably expressing ZNF677 and control cells were transiently transfected with pGL3-Basic or luciferase reporter constructs containing various lengths of the promoter region of CDKN3 (F1: 1408/þ20; F2: 1408/400; F3: 419/188; F4: 210/þ20; left) and HSPB1 (F1: 1298/þ32; F2: 390/þ32; F3: 1232/371; right) genes. Cotransfection with empty vector was used as control. The ratio of the Luc/Renilla activity is shown as mean SD of three independent assays. C, C643 and K1 cells transfected with the indicated constructs and were subjected to ChIP-qPCR assays using anti-Myc tag antibody. Three regions (P1: 1005/810; P2: 419/253; P3: 118/þ20) within CDKN3 promoter, and two regions (P1: 390/198; P2: 315/118) within HSPB1 promoter were selected for ChIP-qPCR assays, respectively. Fold enrichment is shown as mean SD of three independent assays. Statistically significant differences were indicated: , P <0.01; , P <0.001.

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Collectively, these data suggest that ZNF677 functions as a tumor ZNF677. Next, we used the TCGA dataset to investigate the suppressor at least partially through transcriptionally repressing association of ZNF677 expression with the expression of CDKN3 and HSPB1. CDKN3 and HSPB1 in a large cohort of PTCs. Expectedly, there was a significantly negative association between ZNF677 CDKN3 and HSPB1 are identified as downstream targets of expression and the expression of CDKN3 (r ¼0.27; P ZNF677 <0.0001) and HSPB1 (r ¼0.4; P <0.0001; Fig. 4A). Similar By analyzing their promoter sequences using the websites resultswerealsofoundintheLUSCandBRCA(Supplementary (http://genome.ucsc.edu/index.html and http://alternate.meme- Fig. S8). These results suggest that ZNF677 is a transcription suite.org), we found the GCCC(n)T motif (classical zinc finger factor of CDKN3 and HSPB1, and transcriptionally represses binding motif) in the promoters of CDKN3 and HSPB1. We thus their expression via directly binding to the core sequence within suppose that CDKN3 and HSPB1 may be the potential targets of their promoters.

Figure 5. The mechanisms of CDKN3 and HSPB1 regulating Akt phosphorylation. A, K1 and BCPAP cells were transfected with the indicated constructs, and were subjected to Western blot analysis to determine the effect of CDKN3 on the expression of SKP2 and phosphorylation of CDK2 Thr160 using the antibodies against SKP2, CDK2, and phospho-CDK2 Thr160. GAPDH was used as loading control. K1 and BCPAP cells were transfected with pcDNA3.1-CDKN3 or empty vector. CDK2 (B; left), Akt (B; right), and SKP2 (C) were then immunoprecipitated, respectively, and expression of the indicated proteins was evaluated by immunoblotting using corresponding antibodies. D, K1 and BCPAP cells were transfected with pcDNA3.1-CDKN3 or empty vector and were treated with MG132 for 4 h. p21 (left) and Akt (right) were then immunoprecipitated, respectively, and the levels of p21 and Akt ubiquitination were determined by immunoblotting using an anti-ubiquitin (Ub) antibody. E, K1 and FTC133 cells were transfected with the indicated siRNAs and were subjected to Western blot analysis to determine the effect of HSPB1 on Akt phosphorylation. GAPDH was used as loading control. F, K1 and FTC133 cells were transfected with the indicated siRNAs. To determine the interaction between MK2 and Akt, Akt (left) and MK2 (right) were then immunoprecipitated, respectively, and expression of the indicated proteinswas evaluated by immunoblotting using corresponding antibodies.

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ZNF677 Is a Tumor Suppressor in Thyroid Cancer

Next, we cloned their promoters into a pGL3-Basic luciferase tion between SCFSkp2 complex and Akt through preventing its plasmid to construct luciferase reporter plasmids, which were interaction with CDK2/cyclin E (or cyclin A)/p21 (or p27) named as pGL3-CDKN3-Luc (F1: 1408/þ20 bp) and pGL3- complex via CDK2 inactivation. To verify this, we first deter- HSPB1-Luc (F1: 1298/þ32 bp). The results showed that ectopic mined the effect of CDKN3 on CDK2 activity in K1 and BCPAP expression of ZNF677 significantly decreased promoter activity of cells. As expected, ectopic expression of CDKN3 dramatically CDKN3 and HSPB1 in C643 cells relative to the control (Fig. 4B). downregulated phosphorylation of CDK2 at Thr160, while did To identify core region within the CDKN3 and HSPB1 promoters, not affect the expression of CDK2 and SKP2 (Fig. 5A; Supple- three different lengths of CDKN3 promoter region (F2: 1408/ mentary Fig. S10A). Next, we performed a series of reciprocal 400 bp; F3: 419/188 bp; F4: 210/þ20 bp) and two co-IPassaystodeterminetheinteractionsofSKP2withCDK2 different lengths of HSPB1 promoter region (F2: 390/þ32 bp and Akt in K1 and BCPAP cells. As shown in Fig. 5B and C and and F3: 1232/371 bp) were inserted into the pGL3-Basic Supplementary Fig. S10B and S10C, ectopic expression of luciferase plasmid, and cotransfected into C643 cells with CDKN3 attenuated the interaction of SKP2 with CDK2, while pcDNA3.1 (-) A-ZNF677 or empty vector, respectively. The results enhanced its interaction with Akt. Given that both p21 and Akt indicated that pGL3-CDKN3-Luc-F3 and pGL3-HSPB1-Luc-F2 are ubiquitination substrates of SCFSkp2 complex, we next exhibited a significant decrease of luciferase activity in the determined the effect of CDKN3 on ubiquitination of these ZNF677-transfected cells compared with vector-transfected cells, two proteins. The results showed that ectopic expression of but not pGL3-CDKN3-Luc-F2 and -F4, and pGL3-HSPB1-Luc-F3 CDKN3 expectedly decreased p21 ubiquitination, while pro- (Fig. 4B). In addition, similar results were also found in HEK293T moted Akt ubiquitination (Fig. 5D and Supplementary Fig. cells (Supplementary Fig. S9). Taken together, our data suggest S10D), indicating that CDK2 and Akt competitively bind to that ZNF677 may be a potential transcription factor of CDKN3 SKP2. Altogether, our data show that CDKN3 promotes ubi- and HSPB1, and the regulation sites of ZNF677 are located at quitination and subsequent phosphorylation of Akt through 419/188 of CDKN3 gene, and 390/þ32 of HSPB1 gene, preventing the interaction of SKP2 with CDK2 via CDK2 respectively. inactivation. To further determine direct regulation of CDKN3 and HSPB1 HSPB1 is also an important member of heat shock protein by ZNF677 through binding to their promoters, we performed family, and has been characterized as a molecular chaperone the ChIP assay in C643 and K1 cells stably expressing ZNF677 (32). There is evidence showing that HSPB1 regulates Akt and control cells using anti-Myc tag antibody, followed by phosphorylation at Ser-473 through scaffolding the MAPK- qPCR targeting their promoter regions. As shown in Fig. 4C, activated protein kinase-2 (MK2) to Akt signal complex (25). ZNF677 strongly bound to the promoter of CDKN3 or HSPB1 To validate this, we knocked down HSPB1 in K1 and FTC133 in both C643 and K1 cells. Of three different fragments within cells, and evaluated its effect on Akt phosphorylation. As CDKN3 promoter, two fragments (P2: 419/253; P3: 118/ expected, HSPB1 depletion markedly decreased phosphoryla- þ20) were significantly enriched in the ZNF677-transfected tion of Akt Ser473 in these cells relative to the controls (Fig. 5E C643 and K1 cells expressing ZNF677 relative to control cells. and Supplementary Fig. S10E). In addition, the reciprocal co-IP Similarly, two fragments (P1: 390/198; P2: 315/118) assays revealed that HSPB1 knockdown attenuated the inter- within HSPB1 promoter were also enriched in the ZNF677- action between Akt and MK2 in both K1 and FTC133 cells (Fig. transfected C643 and K1 cells expressing ZNF677 relative to 5F and Supplementary Fig. S10F). These findings show that control cells (Fig. 4C). To be consistent with the dual luciferase HSPB1 functions as a scaffold protein to regulate phosphory- findings, the ChIP assays further support CDKN3 and HSPB1 as lation and activation of Akt. direct targets of ZNF677. ZNF677 inhibits tumor growth in nude mice CDKN3 and HSPB1 promote Akt phosphorylation through We also evaluated in vivo tumor-inhibitory effect of ZNF677 distinct mechanisms in nude mice. As shown in Fig. 6A, the tumors induced by K1 Evidently, SCFSkp2 complex directly binds and ubiquitinates cells stably expressing ZNF677 showed significantly longer Akt, ultimately resulting in its phosphorylation and activation latency and smaller mean tumor volume than the tumors (26). SCFSkp2/Cks1 complex also interacts with cyclin-depen- induced by control cells. At the end of the experiments, the dent kinase 2 (CDK2)/cyclin E (or cyclin A) complex to rec- xenograft tumors were isolated and weighed. The mean weight ognize and ubiquitinate p21 or p27 (27–29). The active CDK2 of the tumors stably expressing ZNF677 was significantly less is required for this process. CDKN3, a dual specificity proteins relative to control tumors (P ¼ 0.0009; Fig. 6B). Conversely, the phosphatase, has been shown to interact with, and depho- tumors induced by K1 cells stably knocking down ZNF677 grew sphorylates CDK2 at Thr160, leading to its inactivation (30, more rapidly and showed a significant increase in tumor 31). Thus, we hypothesize that CDKN3 enhances the interac- volume and weight compared with control tumors (Fig. 6C

Figure 6. ZNF677 inhibits xenograft tumor growth. A, Tumor growth curves were compared between K1 cells stably expressing ZNF677 and control cells in nude mice. Data are shown as mean SD (n ¼ 6/group). Day 0 represents time point of tumor cell injection. B, Photographs of dissected tumors from nude mice are presented on the left. Histogram represents mean of tumor weight from ZNF677-overexpressing and control groups (right). C, Growth curves of the tumors induced by K1 cells stably transfected by sh-ZNF677 and sh-NC. Data are shown as mean SD (n ¼ 6/group). Day 0 represents time point of tumor cell injection. D, Left and right panels represent photographs and mean tumor weight of dissected tumors from ZNF677-knockdown and control mice, respectively. Tumor tissues from ZNF677 overexpression and control mice (E) as well as ZNF677-knockdown and control mice (F) were homogenated, lysed, and then subjected to Western blot analysis using the indicated antibodies. GAPDH was used as loading control. Representative tumor sections from ZNF677 overexpression and control mice (G) as well as ZNF677-knockdown and control mice (H) were subjected to IHC staining using the indicated antibodies. Scale bar, 200 mm. Statistically signiﬁcant differences are indicated: , P <0.05; , P <0.01; , P <0.001.

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Figure 7. A schematic model of ZNF677 regulating Akt phosphorylation through transcriptionally repressing its targets CDKN3 and HSPB1.Inthe nucleus, ZNF677 directly binds to the promoter regions of CDKN3 and HSPB1 genes, and represses their transcription. In the cytoplasm, CDKN3 dephosphorylates CDK2 at Thr160, resulting in its inactivation. This prevents SCFSkp2 complex interacting with CDK2/cyclin E (or cyclin A)/p21 complex, thereby enhancing its interaction with Akt and ultimately resulting in phosphorylation and activation of Akt. In addition, HSPB1 functions as a scaffold protein to promote Akt phosphorylation through scaffolding MK2 to Akt signal complex. Collectively, ZNF677 inhibits Akt phosphorylation and activates p53/ p21 signaling through transcriptionally repressing its targets CDKN3 and HSPB1 via distinct mechanisms in thyroid tumorigenesis.

and D). In addition, our results showed that the percentage of Discussion Ki-67–positive cells was significantly decreased in the tumors The KRAB domains are widely present in human zinc finger stably expressing ZNF677, whereas that was dramatically proteins, and considered as potent transcriptional repression increased in the tumors stably knocking down ZNF677 relative modules through interacting with KAP1 and subsequently recruit- to control tumors (Supplementary Fig. S11). ing histone-modifying proteins (11, 33, 34). ZNF677, a member Next, to further confirm the effect of ZNF677 on Akt phos- of KRAB zinc finger protein family, is only reported to be down- phorylation via transcriptionally repressing CDKN3 and HSPB1 regulated by promoter methylation in NSCLC (12). However, its in vivo, Western blot analysis was performed in the xenograft role and exact mechanism in human cancers including thyroid tumors using the indicated antibodies. As shown in Fig. 6E and cancer still has not been elucidated until now. In this study, we Supplementary Fig. S12A, ZNF677 expression was significantly provided strong evidences supporting that ZNF677 is a potent higher in the ZNF677-overexpressing tumors than control tumor suppressor in thyroid cancer. First, we demonstrated that tumors, while the expression of its targets CDKN3 and HSPB1 ZNF677 was frequently downregulated in PTCs compared with was significantly decreased in the former relative to the latter. matched noncancerous tissues, and found the association of As expected, the phosphorylation of Akt Ser473 was dramati- decreased expression of ZNF677 with poor patient outcomes. cally decreased, while p53 expression was upregulated in the Moreover, its inactivation by promoter methylation was con- tumors stably expressing ZNF677 relative to control tumors. firmed by MSP assays and demethylation treatment. Second, On the other hand, as shown in Fig. 6F and Supplementary Fig. ZNF677 reexpression in thyroid cancer cells showed significant S12B, ZNF677 expression was significantly decreased in the growth-inhibitory effect by inhibition of cell proliferation, colony ZNF677-knockdown tumors relative to control tumors. Accord- formation, migration, invasion, and tumorigenic potential in ingly, the levels of CDKN3, HSPB1, and phosphorylated Akt nude mice, and induction of cell-cycle arrest and apoptosis. (Ser473) were increased, while p53 expression was decreased in Conversely, ZNF677 depletion significantly enhanced thyroid the ZNF677-knockdown tumors compared with control cancer cell proliferation and colony formation, further supporting tumors. These data were also supported by IHC assays (Fig. its tumor suppressor function. 6G and H; Supplementary Fig. S12C and S12D). To better understand tumor suppressor activity of ZNF677, Altogether, based on the above findings, we propose a model to we firstevaluateditseffectonaberrantsignalingofthePI3K/ explore molecular mechanisms of ZNF677 inhibiting thyroid Akt and MAPK/Erk pathways in thyroid cancer cells. These two tumorigenesis (Fig. 7). In the nucleus, ZNF677 binds to the pathways play an extensive role in thyroid tumorigenesis, and promoters of its targets CDKN3 and HSPB1, and represses their are major therapeutic targets for this cancer (20). Our results transcription. This results in inhibition of Akt phosphorylation showed that ZNF677 significantly inhibited phosphorylation through distinct mechanisms, and subsequent activation of p53 of Akt Ser473,butnotAktThr308 and Erk in thyroid cancer cells. signaling pathway.

5226 Cancer Res; 78(18) September 15, 2018 Cancer Research

ZNF677 Is a Tumor Suppressor in Thyroid Cancer

It is the fact that the Akt/MDM2/p53 signaling axis plays a assays revealed that CDKN3 expectedly attenuated SKP2 inter- critical role in the regulation of cell cycle and apoptosis in acting with CDK2, while enhanced its interaction with Akt. tumorigenesis (22, 35). In this study, we expectedly found that Accordingly, CDKN3 decreased p21 ubiquitination, while ZNF677 increased the expression of p53 and its downstream increased Akt ubiquitination. These data further support that target p21 in wild-type p53 thyroid cancer cells. However, we CDKN3 promotes ubiquitination and subsequent phosphory- only found that ZNF677 increased the expression of p21, but lation of Akt through preventing the interaction between SKP2 not p53, in mutated p53 cells. These findings suggest that and CDK2 via dephosphorylation and inactivation of CDK2 in ZNF677 induces G0–G1 phase arrest through regulating p21 thyroid cancer cells. In addition, there is evidence showing that via p53-dependent and independent pathways in thyroid can- HSPB1 is a scaffold protein, which promotes phosphorylation cer cells. Given that ZNF677 possesses transcription factor of Akt Ser473 through scaffolding MK2 to Akt signal complex activity, we thus consider that identification of its downstream (25). In this study, we demonstrated that knocking down targets is required to explore the mechanism underlying its HSPB1 in thyroid cancer cells significantly decreased phosphor- regulation on Akt phosphorylation. In fact, a number of genes ylation of Akt Ser473 compared with the controls, and found have been identified to be regulated by ZNF677 in NSCLC (12). that HSPB1 depletion expectedly attenuated the interaction Of them, CDKN3 and HSPB1 have been demonstrated to between Akt and MK2, further supporting that HSPB1 acts as promote Akt phosphorylation (23–25). In this study, we found a scaffold protein to modulate Akt phosphorylation. that the expression of CDKN3 and HSPB1 were negatively In summary, we find that ZNF677 is frequently downregulated regulated by ZNF677 in thyroid cancer cells, and demonstrated by promoter methylation in thyroid cancer, and demonstrate that their promoting effect on Akt phosphorylation and their inhib- ZNF677 is a potent tumor suppressor through a series of in vitro itory effect on p53/p21 signaling. In addition, we also identi- and in vivo experiments. Mechanistically, ZNF677 plays its tumor fied that these two genes were direct targets of ZNF677 by a suppressor role in thyroid cancer through transcriptionally repres- series of luciferase and ChIP-qPCR assays, and demonstrated sing its downstream targets CDKN3 and HSPB1, and thereby that ZNF677 inhibited Akt phosphorylation and activated p53/ inhibiting phosphorylation and activation of Akt. p21 signaling in thyroid cancer cells at least partially through transcriptionally repressing its targets CDKN3 and HSPB1. Disclosure of Potential Conflicts of Interest Although CDKN3 has been reported to enhance Akt phos- No potential conflicts of interest were disclosed. phorylation (23), its exact mechanism remains totally unclear. It is well known that Skp2 is the substrate-recruiting compo- Authors' Contributions nent of the SCFSkp2 complex, which plays a critical role in cell- Conception and design: Y. Li, M. Ji, P. Hou cycle regulation and tumorigenesis by promoting K48 ubiqui- Development of methodology: Y. Li, Q. Yang tination and degradation of p21 and p27 (27–29). SKP2 Acquisition of data (provided animals, acquired and managed patients, binding to active CDK2/cyclin E (or cyclin A) complex is provided facilities, etc.): H. Guan, B. Shi required to recruit and recognize its substrates such as p21 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, fi computational analysis): Y. Li, Q. Yang and p27 (36). CDKN3 is a dual speci city proteins phospha- Writing, review, and/or revision of the manuscript: Y. Li, M. Ji, P. Hou fi tase, and is identi ed as a cyclin-dependent kinase inhibitor Study supervision: P. Hou to dephosphorylate and inactivate CDK2 (30, 31). These observations suggest that CDKN3 prevents SCFSkp2 complex bind- Acknowledgments ing to CDK2, thereby inhibiting ubiquitination and degrada- We would like to thank Drs. Haixia Guan (The First Affiliated Hospital of tion of p21 and p27 through dephosphorylating and inactivat- China Medical University, Shenyang, China) and Lei Ye (Ruijin Hospital, ing CDK2. Moreover, there is increasing evidence showing that Shanghai, China) for kindly providing human thyroid cancer cell lines. This SCFSkp2 complex directly ubiquitinates and subsequently work was supported by the National Natural Science Foundation of China (no. phosphorylate Akt, thereby resulting in its activation (26). 81372217, 81572627, and 81672645). Given the above observations, we hypothesize that CDKN3 The costs of publication of this article were defrayed in part by the payment of prevents SCFSkp2 complex interacting with CDK2, thereby page charges. This article must therefore be hereby marked advertisement in enhancing its interaction with Akt and ultimately resulting in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. phosphorylation and activation of Akt. In this study, we demonstrated that ectopic expression of CDKN3 significantly Received January 15, 2018; revised May 29, 2018; accepted July 5, 2018; decreased phosphorylation of CDK2 at Thr160, and co-IP published first July 11, 2018.

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5228 Cancer Res; 78(18) September 15, 2018 Cancer Research

ZNF677 Suppresses Akt Phosphorylation and Tumorigenesis in Thyroid Cancer

Yujun Li, Qi Yang, Haixia Guan, et al.

Cancer Res 2018;78:5216-5228. Published OnlineFirst July 11, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-0003

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