Tao et al. Clin Epigenet (2020) 12:173 https://doi.org/10.1186/s13148-020-00959-6

RESEARCH Open Access The tumor suppressor Zinc fnger protein 471 suppresses breast cancer growth and metastasis through inhibiting AKT and Wnt/ β‑catenin signaling Chunfang Tao1, Juan Luo1, Jun Tang1, Danfeng Zhou1, Shujun Feng2, Zhu Qiu1, Thomas C. Putti3, Tingxiu Xiang1, Qiao Tao1,4, Lili Li4* and Guosheng Ren1*

Abstract Background: Zinc-fnger protein 471 (ZNF471) is a member of the Krüppel-associated box domain zinc fnger protein (KRAB-ZFP) family. ZNF471 is methylated in squamous cell carcinomas of tongue, stomach and esophageal. However, its role in breast carcinogenesis remains elusive. Here, we studied its expression, functions, and molecular mechanisms in breast cancer. Methods: We examined ZNF471 expression by RT-PCR and qPCR. Methylation-specifc PCR determined its promoter methylation. Its biological functions and related molecular mechanisms were assessed by CCK-8, clonogenicity, wound healing, Transwell, nude mice tumorigenicity, fow cytometry, BrdU-ELISA, immunohistochemistry and West- ern blot assays. Results: ZNF471 was signifcantly downregulated in breast cell lines and tissues due to its promoter CpG meth- ylation, compared with normal mammary epithelial cells and paired surgical-margin tissues. Ectopic expression of ZNF471 substantially inhibited breast tumor cell growth in vitro and in vivo, arrested cell cycle at S phase, and pro- moted cell apoptosis, as well as suppressed metastasis. Further knockdown of ZNF471 verifed its tumor-suppressive efects. We also found that ZNF471 exerted its tumor-suppressive functions through suppressing epithelial-mesen- chymal transition, tumor cell stemness and AKT and Wnt/β-catenin signaling. Conclusions: ZNF471 functions as a tumor suppressor that was epigenetically inactivated in breast cancer. Its inhibi- tion of AKT and Wnt/β-catenin signaling pathways is one of the mechanisms underlying its anti-cancer efects. Keywords: ZNF471, CpG methylation, Breast, AKT, Wnt signaling

Introduction In women, breast cancer is the most commonly diag- nosed cancer and the leading cause of cancer death *Correspondence: [email protected]; [email protected] worldwide [1, 2]. It is a heterogeneous disease divided 1 Key Laboratory of Molecular Oncology and Epigenetics, The First into fve major subtypes by DNA microarray gene expres- Afliated Hospital of Chongqing Medical University, Chongqing, China 4 Cancer Epigenetics Laboratory, Department of Clinical Oncology, State sion profling: luminal A (estrogen receptor [ER]+, pro- Key Laboratory of Translational Oncology, Sir YK Pao Center for Cancer gesterone receptor [PR]±, HER2−), luminal B (ER+ , and Li Ka Shing Institute of Health Sciences, The Chinese University PR , HER2 ), HER2-enriched (ER , PR , HER2 ), of Hong Kong and CUHK Shenzhen Research Institute, Hong Kong, China ± + − − + Full list of author information is available at the end of the article basal-like (ER−, PR−, HER2−), and normal-like cancers

© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativeco​ mmons​ .org/licen​ ses/by/4.0/​ . The Creative Commons Public Domain Dedication waiver (http://creativeco​ ​ mmons.org/publi​ cdoma​ in/zero/1.0/​ ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tao et al. Clin Epigenet (2020) 12:173 Page 2 of 14

[3, 4]. Currently, the main treatment methods for breast a panel of breast cancer cell lines, normal mammary epi- cancer are surgery, medical oncology (chemotherapy, thelial cell lines (HMEC and HMEpC) and normal breast endocrine therapy, or HER2-directed therapy), and radia- tissues by semiquantitative RT-PCR. ZNF471 was read- tion. Endocrine therapy and HER2 targeted therapies ily detected in HMEpC and HMEC cells, but dramati- have led to a dramatic improvement in the overall prog- cally reduced or silenced in six of nine breast cancer cell nosis of ER/PR+ and HER2+ subtypes, respectively [5]. lines, (Fig. 1a). Data from the Oncomine database (https​ However, the incidence and mortality of breast cancer ://www.oncom​ine.org/) showed that ZNF471 mRNA remain high in many countries. Terefore, understand- expression was downregulated in Invasive Breast Carci- ing the molecular mechanisms involved in breast cancer noma (IBC), Invasive Ductal Breast Carcinoma (IDBC) development is critical. and Invasive Lobular Breast Carcinoma (ILBC) compared Te Wnt/β-catenin pathway participates in the devel- to normal breast tissues (Fig. 1b). Furthermore, ZNF471 opment of various tumors. Wnt signaling can facilitate expression was associated with progesterone receptor tumor metastasis [6] and is important for maintain- (PR), HER2, nodal status and tumor grade of breast can- ing cell stemness [7–9]. Activation of the Wnt pathway cer. Tese data indicated that ZNF471 expression is fre- can be detected in more than half of breast cancers, quently downregulated in breast cancer and associated and patients with Wnt pathway activation tend to have with clinicopathologic features including PR, HER2 sta- worse clinical outcomes [10]. Te AKT pathway has been tus, lymph node metastasis and higher histologic grade shown to be involved in breast cancer cell proliferation, (Fig. 1c, d). To analyze the relationship between ZNF471 apoptosis, and metastasis [11, 12]. and survival in breast cancer, a prognostic analysis was Cancer is driven by accumulated genetic and epigenetic next performed using the Human Protein Atlas database alterations. CpG island hypermethylation, a common (https​://www.prote​inatl​as.org/). Results showed that epigenetic change, has been found in almost all types of patients with higher ZNF471 mRNA expression levels human tumors [13]; thus, methylation might be an ideal had increased survival probability compared to those candidate for early detection of tumors [14]. In breast with low ZNF471 mRNA levels (Fig. 1d). We further per- cancer, promoter hypermethylation has been reported formed the univariate and multivariate Cox regress anal- to be involved in tumor suppressor gene (TSG) silencing yses through analyzing breast cancer genomic data from [15–17]. the TCGA database (n = 639), however,we found that Krüppel-associated box domain zinc fnger proteins ZNF471 is not an independent predictor in breast cancer (KRAB-ZFPs) are the largest family of transcriptional (Additional fle 1: Table S1). Te diference between our regulators in mammals and are characterized by the pres- result and the Human Protein Atlas database result may ence of a C-terminal array of a DNA-binding domain be due to the diferent samples selected. and an N-terminal KRAB domain [18]. KRAB-ZFPs To analyze the relationship between ZNF471 and sur- have been implicated in cell diferentiation, prolifera- vival in breast cancer, a prognostic analysis was next per- tion, apoptosis, neoplastic transformation, and metabolic formed using the Human Protein Atlas database (https​ pathways [18, 19]. KRAB-ZFPs have also been linked to ://www.prote​inatl​as.org/). Results showed that patients cancer development [20–23]. ZNF471 is a member of the with higher ZNF471 mRNA expression levels had KRAB-ZFP family, located in 19q13.43. It has been iden- increased survival probability compared to those with tifed as a TSG in gastric cancer where it directly inhib- low ZNF471 mRNA levels (Fig. 1d). its transcription of its downstream targets TFAP2A and PLS3 [24]. In esophageal cancer, ZNF471 functions as a Promoter methylation mediates ZNF471 downregulation TSG through activating MAPK10/JNK3 signaling [25]. in breast cancer However, the role of ZNF471 in breast cancer remains We next examined whether ZNF471 downregula- unclear. In this study, we showed that ZNF471 was tion in breast cancer was due to promoter methyla- silenced or downregulated in breast cancer due to pro- tion. ZNF471 was methylated in 4 of 7 breast cancer moter methylation. We also showed that ZNF471 exerted cell lines (Fig. 1a). A pharmacological demethylation its tumor-suppressive functions through AKT/GSK-3β experiment was performed in which MDA-MB-231, and Wnt/β-catenin signaling pathways. YCC-B1 and MCF-7 cells were treated with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine Results (Aza) alone or in combination with the HDAC inhibitor ZNF471 downregulation in breast cancer is associated trichostatin A (TSA). Te results indicated that phar- with poor patient survival macologic demethylation partially restored the expres- To assess whether ZNF471 is downregulated in breast sion of ZNF471, along with decreased methylated tumors, we frst examined the expression of ZNF471 in alleles and increased unmethylated alleles as detected Tao et al. Clin Epigenet (2020) 12:173 Page 3 of 14

Breast Ca 6 -3 3 a 1 C

1 8 b 9 R B Breast cancer (TCGA) Curtis Breast 54 O CF-7 T BT00 2 47 D Marker s HMEC HMEp B H N NBT010 NBT014 MB23 MB46 M SK-B T YCC-B1 YCC- ZR-75-

ZNF471

GAPDH 3 1 - 1 s 1 7 9 5- 4 BR 7 F- - 5 - O K 2 Marker BT MB23 MC T47D YCC-B ZR H S M MSP U

c d Nodalstatus Scarff-Bloom-Richardson(SBR) Breast cancer (TCGA) el l l v e 10 e PR (IHC) HER2 (IHC) v

vel 6 le v 10 > e e le p=0.029 < > 9 Al Al 4 NA N N 4

2 R

R 8 mRNA ty i mR 2

dm 5 d dm 2 7 ze ze ze i

0 di d

r 0 6 r a a ardi d d d 0 Probabil n 5 n

a 0 -2 standardized ta -2 st stan 4 g2 2

2s -2 o g2 -4 g g l o o l l 1 lo 3 7 1 1

-5 Survival -4 -4 7 7

4 -6 2 4 p=0.0456 p=0.0007 F F471 F p<0.0001 p<0.0001 ZNF4 N N ZNF471 low expression (n=277) Z

Z 1 ZN HER2-HER2+ PR-PR+ ZNF471 high expression (n=798) (n=1343) (n=1975) (n=2336) (n=436) N- N+ SBR1 SBR2 SBR3 0 (n=4248) (n=3336) (n=849)(n=2813)(n=2791) 02 46810121416182023 Time (years) Fig. 1 Expression of ZNF471 in breast cancer and its association with clinicopathologic features of breast tumors and survival probability. a ZNF471 expression levels were examined in nine breast cancer cells, normal mammary epithelial cell lines and normal breast tissues by RT-PCR, NBT: normal breast tissue. The promoter methylation levels of ZNF471 in breast cancer cells. b ZNF471 expression in breast cancer from the Oncomine database (https​://www.oncom​ine.org/). IBC: Invasive Breast Carcinoma; IDBC: Invasive Ductal Breast Carcinoma; ILBC: Invasive Lobular Breast Carcinoma (ILBC). c ZNF471 mRNA expression was associated with PR, HER2 status in breast cancer. (https​://bcgen​ex.centr​egaud​uchea​u.fr/BC-GEM/GEM-Accue​ il.php?js 1). d ZNF471 mRNA expression was related to lymph node metastasis, SBR grade and survival probability in breast cancer =

by methylation-specifc PCR (MSP) (Fig. 2a, b). High- ZNF471 inhibits breast tumor cell growth and colony resolution bisulfte genomic sequencing (BGS) analysis formation was performed to examine the methylation status of To clarify the efect of ZNF471 in breast cancer, we frst 43 individual CpG sites within the ZNF471 promoter established cell lines that stably overexpress ZNF471. CGI, with a higher density of methylated alleles were MDA-MB-231 and YCC-B1 cells were transfected with observed in methylated MB231 and YCCB1 cell lines empty pcDNA3.1 and ZNF471 and selected with G418 compared with HMEC cell lines, consistent with the for 14 days. Expression levels of ZNF471 in the trans- MSP results (Fig. 2c). fected cells were examined by RT-PCR, qPCR, and west- Moreover, ZNF471 methylation was detected in ern blotting (Fig. 3a–c).Colony formation and CCK-8 53/64 (82.8%) tumors but only 1/16 (6.25%) in breast proliferation assays were performed to assess the efect of normal tissues (Fig. 2d, e; Additional fle 2: Fig. S1). ZNF471 on cell proliferation in breast cancer. Te CCK-8 Consistently, the mRNA level of ZNF471 was sig- assay showed that cell viability was decreased at 24, 48, nifcantly lower in 12 of 14 pairs of breast cancer tis- and 72 h in ZNF471-expressing cells (Fig. 3d). A reduc- sues compared with paired surgical-margins (Fig. 2f). tion of approximately 55–80% in colony formation was Online databases were further used to investigate observed in ZNF471-expressing cells compared to con- whether ZNF471 downregulation in breast cancer was trol cells (Fig. 3e). Tese results indicated that ZNF471 related to promoter methylation (https​://methh​c.mbc. suppressed cell colony growth and colony formation in nctu.edu.tw/). Results showed that ZNF471 methyla- breast cancer. tion was far more prevalent in breast cancer tissues than in normal breast tissues, and downregulation of ZNF471 induces S cell cycle arrest and induces apoptosis ZNF471 in breast cancer was signifcantly inversely in breast cancer correlated with its methylation (Fig. 2g, h). Tese data We next examined the role of ZNF471 in cell cycle indicated that ZNF471 was downregulated in breast distribution and apoptosis via flow cytometry. There cancer due to promoter methylation. was an increase in the number of S phase cells in Tao et al. Clin Epigenet (2020) 12:173 Page 4 of 14

Breast tumortissues s r

a e

e k r Ma MSP-M arkers M MSP-M Marker s MSP-M s

MB231 YCCB1 MCF7 Marker MSP-M b c m44 m6 f MB231 MCF-7 CpGsite(#): T T

+ HMEC A A+ ZNF471

GAPDH MB231

M MSP U YCCB1

g p<0.001 h r Breast Invasive Ca (TCGA) e

ot 0.7 ) d m p<0.00001 o 0.6 (% r promoter

p r=-0.607 n 0.5 o ti 71 a 4 0.4 F

N 0.3 ZNF471 thyl Z f 0.2 of me no 0.1 71 on i 4 t 0

ZN F -0.1 hyla t -0.2 Methylatio

Me -6 -3 03 BN BC BN BC ZNF471 expression (Foldchange, log2) n=92 n=735 n=92 n=92 Fig. 2 ZNF471 is downregulated in breast cancer cell lines and tissues due to promoter methylation. a, b Pharmacological demethylation restored the expression of ZNF471 in breast cancer cell lines, with demethylation of the promoter. M, methylated; U, unmethylated. c High-resolution methylation analysis of ZNF471 promoter by BGS in HMEC, MB231 and YCCB1 cells. ZNF471 promoter methylation levels were detected in breast normal tissues (d) and breast cancer tissues (e). f ZNF471 mRNA expression in primary breast tumor tissues (n 14) and paired adjacent = non-cancerous tissues (n 14) by qPCR. g ZNF471 promoter methylation in breast cancer tissues and normal breast tissues from an online database = (https​://methh​c.mbc.nctu.edu.tw/). BN: breast normal tissues; BC: breast cancer tissues. h An online database was used to analyze the correlation between the expression of ZNF471 and its promoter methylation in breast cancer (https​://methh​c.mbc.nctu.edu.tw/). Error bars mean standard deviation (SD); values are presented as the mean SD of at least three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001) ±

ZNF471-transfected MD-MB-231 and YCC-B1 cells ZNF471 suppresses breast tumor cell migration compared to empty-transfected cells (Fig. 4a). Fur- and invasion thermore, the BrdU-ELISA assay, which reflects active To evaluate the efect of ZNF471 on cell migration and DNA synthesis, revealed that cell proliferation was invasion, wound healing and Transwell assays were per- decreased in ZNF471-expressing cells (Fig. 4b). Flow formed. Te wound-healing assay showed that the rate cytometric analysis also revealed that ZNF471 overex- of healing in ZNF471-overexpressing cells was signif- pression dramatically induced apoptosis compared to cantly slower than that in control cells at 24 and 48 h the control (Fig. 4c). We next assessed the expression (Fig. 5a). Furthermore, the number of ZNF471-overex- of the following apoptotic markers by western blot- pressing cells that passed through the chamber mem- ting: cleaved-caspase 9, cleaved-PARP, Bax, Bcl-2, and brane was decreased compared to cells transfected with p53. Expression of cleaved-caspase 9, cleaved-PARP, the empty control vector in the Transwell migration Bax and p53 was increased, while the expression of assay (Fig. 5b). Tese data demonstrated that ZNF471 Bcl-2 was decreased, further confirming the effect of inhibited breast cancer cell migration. A Matrigel inva- ZNF471 on apoptosis (Fig. 4d). Taken together, these sion assay revealed that the number of cells passing data showed that ZNF471 blocked the cell cycle in the through the chamber membrane with a Matrigel barrier S phase and inducing apoptosis. was lower than control cells (Fig. 5c). Tus, ZNF471 Tao et al. Clin Epigenet (2020) 12:173 Page 5 of 14

a MB231 YCCB1 1 r s

7 d o ct Ve Vector Marker ZNF4 ZNF471

ZNF471 194 bp

GAPDH 206bp

b

e Vector ZNF471 1 MB231 YCCB1 MB23

c MB231 YCCB1 Vector ZNF471 Vector ZNF471 ZNF471 73 kDa YCCB1 GAPDH 37 kDa MB231YCCB1 Fig. 3 ZNF471 inhibits breast cancer cell growth and colony formation. Ectopic expression of ZNF471 in MB231 and YCCB1 cells was measured by RT-PCR (a), qRT-PCR (b) and Western blot assay (c). d Cell viabilities were measured with CCK-8 assay at 24, 48 and 72 h in vector- and ZNF471-stably transfected MB231 and YCCB1 cells. e Cell colony formation ability was evaluated by clonogenicity in vector- and ZNF471-expressing MB231 and YCCB1 cells. Left: Representative images. Right: Data summary. All values are presented as the mean SD of at least three independent ± experiments. SD, standard deviation. **p < 0.01; ***p < 0.001

can suppress cell invasion. Tese results indicated that ZNF471 attenuates the epithelial‑mesenchymal transition ZNF471 inhibits cell migration and invasion. (EMT) and suppresses cell stemness in breast cancer Because the EMT plays an important role in cell migra- tion and invasion by reducing cell–cell adhesion and Knockdown of ZNF471 promotes cell growth, induces increasing motility, we frst tested whether ZNF471 metastasis and inhibits apoptosis inhibits breast cancer cell migration and invasion by To further demonstrate the tumor suppression func- attenuating the EMT. Morphological changes of cells tion of ZNF471 in breast cancer, we knockdown of stably expressing ZNF471 and empty vector cells were ZNF471 in ZNF471-expressing breast cancer cell line observed by phase-contrast microscopy. We found that BT549 by siRNA transfection. Western blot assay was the morphology of ZNF471-expressing cells dramati- used to detect the knockdown efciency of ZNF471 cally changed from a spindle-like morphology to a typi- (Fig. 6a). In the CCK-8 assay, we found that cell viability cal cobblestone-like morphology (Fig. 7a). Western blot was increased after ZNF471 knocking down (Fig. 6b). was then performed to detect the expression of EMT Cell migration and invasion abilities were measured by markers, the results revealed that the epithelial marker Transwell assays, results indicated that knockdown of E-cadherin was upregulated while Vimentin, Snail, Slug, ZNF471 induced cell migration and invasion compared and N-cadherin were downregulated. Metastasis markers with cells transfected with control siRNA (Fig. 6c). matrix metalloproteinase MMP1 and MMP3 were also Moreover, fow cytometry assay and western blotting downregulated in ZNF471-overexpressing cells (Fig. 7b). demonstrated that knockdown of ZNF471 inhibited cell However, the expression of EMT markers and MMP1 apoptosis (Fig. 6d, e). Tese knockdown data confrmed was upregulated after ZNF471 knocking down (Fig. 7c). that ZNF471 has a tumor-suppressive efect on breast Tus, ZNF471 suppressed breast cancer cell metastasis cancer. by attenuating the EMT. Tao et al. Clin Epigenet (2020) 12:173 Page 6 of 14

a Vector ZNF471

G1:67.83% G1:59.87% b G2:5.51% G2:6.41% S:26.66% S:33.71% 23 1 MB

G1:68.99% G1:68.55% G2:3.59%

1 G2:16.17% S:14.84% S:27.86% YCCB

c Vector ZNF471 d MB231 YCCB1

1 Vector ZNF471 Vector ZNF471 Cleaved-Casp9 17 kDa MB23 Cleaved-PARP 89 kDa

Bax 20 kDa

Bcl-2 26 kDa

p53 53 kDa

YCCB 1 GAPDH 37 kDa

Fig. 4 ZNF471 induces cell cycle at S phase and induces apoptosis. a The cell cycle distribution was measured by fow cytometry analysis in vector- and ZNF471-expressing MB231 and YCCB1 cells. Left: Representative fow cytometry plots. Right: Data summary. b BrdU-ELISA assay at 24 h in vector- and ZNF471-transfected MB231 and YCCB1 cells. c The percentages of apoptotic cells were evaluated in vector- and ZNF471-transfected MB231 and YCCB1 cells. Left: Representative fow cytometry plots. Right: Quantitative analysis. d Western blot analysis of apoptotic markers (cleaved-caspase 9, cleaved-PARP, Bax, Bcl-2, and p53). All values are presented as the mean SD of at least three independent experiments. SD, ± standard deviation. **p < 0.01; ***p < 0.001

As the EMT is closely related to cancer cell stemness, (Fig. 8a–c). Immunohistochemistry and hematoxylin and we next verifed whether ZNF471 afects cell stemness. eosin-stained sections showed that expression of Ki-67, a We analyzed stem cell markers (ABCG2, BMI1, KLF4, marker of cell proliferation, was signifcantly decreased, MAD2, STAT3, CD44, MYC, NANOG2, and OCT4) by and the number of tumor cells exhibiting nuclear frag- qPCR. We found that expression of ZNF471 resulted in mentation was increased (Fig. 8d). Tese results suggest the downregulation of almost all of the stem cell mark- that ZNF471 inhibits breast cancer growth in vivo. ers in MDA-MB-231 and YCC-B1 cells (Fig. 7d). Tus, ZNF471 suppressed cell stemness in breast cancer. ZNF471 exerts its tumor‑suppressive function by blocking AKT and Wnt/β‑catenin signaling pathways ZNF471 inhibits breast tumor growth in vivo AKT and Wnt/β-catenin pathways have been reported Given that ZNF471 suppressed breast cancer cell pro- to be associated with breast cancer cell proliferation and liferation in vitro, we further tested its function in vivo. metastasis. We assumed that the anti-cancer efect of Stable MDA-MB-231 cells transfected with ZNF471 ZNF471 in breast cancer was achieved by modulating and empty pcDNA3.1 were injected into nude mice. AKT and Wnt/β-catenin signaling. Terefore, expres- Tumor length and width were measured every 2 days. sion levels of key proteins of the AKT and Wnt/β-catenin Tumors were derived from mice after 21 days for fur- pathways were investigated by western blotting. Te 2 ther analysis. Tumor volume (length × width ) and results showed that ectopic expression of ZNF471 down- average weight were signifcantly lower in cells with sta- regulated expression of phospho-AKT, phospho-GSK-3β, ble expression of ZNF471 than that in control tumors active-β-catenin, and the following downstream target Tao et al. Clin Epigenet (2020) 12:173 Page 7 of 14

Vector a ZNF471

Vector ZNF471

b c Vector Vector ZNF471 ZNF471 1 31 2 MB23 n MB 1 Invasion Migratio YCCB 1 YCCB

Fig. 5 ZNF471 suppresses migration and invasion of breast tumor cells. a Wound healing assay in vector- and ZNF471-transfected MB231 and YCCB1 cells at 0, 24, and 48 h. Left: Representative images. Right: Quantitative analysis of the wound healing rate. b Transwell assay showed the migration ability of vector- and ZNF471-expressing MB231 and YCCB1 cells Left: Representative images. Right: Data summary. c Transwell assay showed the invasion ability of vector- and ZNF471-expressing MB231 and YCCB1 cells. Left: Representative images. Right: Data summary. Data are presented as the mean SD. **p < 0.01; ***p < 0.001 ± genes: c-Myc, cyclin D1 (Fig. 8e). In contrast, knockdown committee of the frst afliated hospital of Chongqing of ZNF471 in BT549 cells by siRNA upregulated expres- Medical University, and all patients signed consent forms. sion of them. Collectively, ZNF471 was a negative regula- tor of AKT and Wnt/β-catenin pathways and exerted its anti-cancer efect by inhibiting the activation of AKT and RNA isolation, reverse transcription‑PCR, semiquantitative Wnt/β-catenin signaling. RT‑PCR, and quantitative real‑time PCR Total RNA was extracted from cell lines and tissues Methods and materials using TRIzol reagent (Invitrogen). RNA was reverse- Tumor cell lines and tissues transcribed according to the instructions of the Reverse A panel of breast cancer cell lines (MDA-MB-231, MDA- Transcription System (Promega, Madison, WI, USA). MB-468, BT549, MCF-7, T47D, SK-BR-3, YYC-B1, YCC- RT-PCR was carried out with Go-Taq DNA polymerase B3, and ZR-75–1) was used. Human mammary epithelial (Promega) and performed using a fnal volume of 10 μL cell lines HMEpC and HMEC were used as controls. All reaction mixture containing 2 μL cDNA for 32 cycles cell lines were maintained at 37 °C in RPMI 1640 with of amplifcation as previously reported [26]. 2% agarose 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, gels were used to assay amplifed PCR products. GAPDH USA). Human normal adult breast tissue RNA samples served as a control. Te qPCR was carried out by Max- were purchased commercially (Stratagene, La Jolla, CA, ima SYBR Green/ROX qPCR MasterMix (MBI Fermen- USA; Millipore Chemicon, Billerica, MA, USA; and Bio- tas, St. Leon-Rot, Germany) on an Applied Biosystems Chain Institute, Hayward, CA, USA). Breast cancer tis- 7500 Real-Time PCR System (Applied Biosystems, Foster sues and paired surgical-margin tissues were obtained City, CA)according to the manufacturer’s instructions from the First Afliated Hospital of Chongqing Medi- [26]. Te relative expression of ZNF471 was calculated cal University. Tis study was approved by the ethics by the 2(− ΔCt) method. GAPDH was used as a control. Tao et al. Clin Epigenet (2020) 12:173 Page 8 of 14

A #C b a iRNA -s 1 7 C N NF471-siRNA# F4 si N Z ZNF471-siRNA#BZ

ZNF471 73 kDa

GAPDH 37 kDa

BT549

c siNC ZNF471-siRNA#A n d e atio siNC ZNF471-siRNA#C

9 BT549 Migr

siNC ZNF471-siRNA#C 9 BT54

54 Cleaved-Casp9 17 kDa BT Cleaved-PARP 89 kDa Invasion p53 BT549 53 kDa

siNC ZNF471-siRNA#C Bcl-2 26 kDa

n ZNF471 73 kDa

GAPDH 37 kDa 9 Migratio BT54 Invasion

Fig. 6 Knockdown of ZNF471 promotes cell growth, metastasis, and inhibits cell apoptosis. a Western blot assay of ZNF471 knockdown efciency in BT549 cells after transfected with. ZNF471. siRNA and control siRNA. b Cell viabilities were measured by CCK-8 assay at 24, 48 and 72 h in BT549 cells after ZNF471 knockdown. c Transwell assay showed cell migration and invasion of BT549 cells after the ZNF471 knockdown. Left: Representative images. Right: Data summary. d Flow cytometry assay of relative cell apoptosis rate in BT549 cells after transfected with ZNF471 siRNA and control siRNA. (E) Western blotting analyzed the expression of cleaved-casppase9, cleaved PARP, Bcl-2, and p53 in ZNF471 knockdown cells. Data are presented as the mean SD. **p < 0.01; ***p < 0.001 ±

All samples were assessed in triplicate. All primers are Construction of vector‑ and ZNF471‑expressing stable cell shown in Table 1. lines MDA-MB-231 and YCC-B1 cells were plated in six-well Methylation‑specifc PCR and bisulfte genomic plates and transfected with pcDNA3.1 and pcDNA3.1– sequencing ZNF471 plasmids at a concentration of 4 µg using Lipo- fectamine 2000 (Invitrogen). At 48 h after transfection, To evaluate the methylation of ZNF471, bisulfte modif- G418 was added to screen cells expressing ZNF471 for cation of DNA, MSP and BGS were performed as previ- 14 days. Stable cells were further verifed by RT-PCR, ously [27, 28]. Genomic DNA was isolated from tissues qPCR, and western blotting. and cell lines using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany).MSP was carried out using AmpliTaq- Cell viability assays Gold DNA Polymerase (Applied Biosystems) with the fol- Vector- and ZNF471-expressing cells were plated in lowing conditions: 95 °C for 10 min and 40 3-step cycles 96-well plates at a density of 2000 cells/well. After (94 °C for 30 s, 58 °C for 30 s and 72 °C for 30 s), a fnal the cells were adherent, Cell Counting Kit-8 (CCK- extension of 5 min at 72 °C. PCR products were assessed 8; Dojindo Molecular Technologies, Inc., Kuma with 2% agarose gels. Te primers are listed in Table 1. - moto, Japan) reagent was added at a concentration of 10 μL/100 μL complete medium to assess viability at 0, 5‑Aza‑2′‑deoxycytidine and trichostatin A treatment 24, 48, and 72 h. Absorbance was read in a microplate Cell lines were treated with 10 mM Aza (Sigma-Aldrich, reader at 450 nm. All experiments were performed in St. Louis, MO, USA) for 3 days followed by treatment triplicate. with or without 100 nM TSA (Cayman Chemical Co., Ann Arbor, MI, USA) for 24 h. Cells were then collected for RNA extractions. Tao et al. Clin Epigenet (2020) 12:173 Page 9 of 14

ZNF471- a Vector ZNF471 c siNC siRNA#C N-cad 140 kDa

1 Vimentin 57 kDa

Snail 29 kDa MB23

Slug 30 kDa

MMP1 52 kDa

GAPDH 37 kDa BT549 b d

Fig. 7 ZNF471 attenuates the epithelial-mesenchymal transition (EMT) and suppresses cell stemness in breast cancer. a Morphological changes of MB231 cells after transfected with vector and ZNF471. b Expression of E-cadherin, N-cadherin, Vimentin, Snail, Slug, and MMP1,3 by western blot assay in empty vector cells and ZNF471 overexpressing cells. c Western blot was performed to examined N-cad, vimentin, slug, snail and MMP1 in BT549 cells. d qPCR analysis of stem cell markers expression (Data are presented as the mean SD; *p < 0.05; **p < 0.01; ***p < 0.001) ±

Colony formation assay washed once with PBS, and incubated with Annexin Stably expressing ZNF471 and empty vector cells were V-fuorescein isothiocyanate (FITC; BD Pharmingen) and seeded in six-well plates at 200, 400, or 800 cells/well in PI (Sigma-Aldrich) at room temperature for 30 min. Te complete medium containing G418. Surviving colonies fow cytometry results were evaluated using a FACSCali- (> 50 cells per colony) were counted after 10–14 days, bur machine (BD Biosciences, Franklin Lakes, NJ, USA). fxed with 4% paraformaldehyde, and stained with crys- Experiments were performed in triplicate. tal violet. All experiments were performed in triplicate. BrdU cell proliferation enzyme‑linked immunosorbent Flow cytometry assay Flow cytometry was performed to evaluate the cell cycle Te BrdU Cell Proliferation ELISA Kit (Abcam, Cam- and apoptosis. For cell cycle analysis, cells stably express- bridge, UK) was used for the detection of DNA synthe- ing ZNF471 and vector control cells were trypsinized sis and cell proliferation. Following transfection with with pancreatin, washed once with phosphate-bufered ZNF471 and pcDNA3.1 plasmids for 48 h, cells were saline (PBS), and fxed in 70% ice-cold ethanol overnight. plated in 96-well plates at 1 × 105 cells/mL in 100 μL/ Propidium iodide (PI) was used to stain cells for 30 min well of complete cell culture media. After 24 h of culture, in the dark. For apoptosis, cells were transfected with bromodeoxyuridine (BrdU) was added into the wells pcDNA3.1 and pcDNA3.1–ZNF471 plasmids at a con- for another 12 h. Te BrdU-ELISA assay was performed centration of 4 μg using Lipofectamine 2000 (Invitro- according to the manufacturer’s instructions. Results gen). After 48 h, cells were trypsinized with pancreatin, were read using a microplate reader at 450 nm. Tao et al. Clin Epigenet (2020) 12:173 Page 10 of 14

a b c Vector p<0.05

ZNF471

Vector

ZNF471

d Vector ZNF471 e MB231 YCCB1 BT549 Vector ZNF471 Vector ZNF471 siNC ZNF471-siRNA#C p-AKT 60 kDa

H&E Total-AKT 60 kDa

p-GSK-3β 47 kDa

active-β-Catenin 92 kDa

Total-β-Catenin 92 kDa

c-Myc 57 kDa

Ki-67 CyclinD1 37 kDa GAPDH 37 kDa

Fig. 8 ZNF471 inhibits breast tumor growth in vivo. ZNF471 exerts its tumor-suppressive function by blocking AKT and Wnt/β-catenin signaling pathways. a Images of human breast tumor xenografts. b Tumor growth curve for vector- and ZNF471-expressing MB231 cells in nude mice xenografts. c Tumor weights in the two groups of nude mice. d Representative photographs of H&E staining and IHC expression analyses of Ki-67. e Expression of phospho-AKT, phospho-GSK-3β, active-β-catenin, and the following downstream target genes: c-Myc, cyclin D1 in MB231, YCCB1 and BT549 cells. Data are presented as the mean SD. *p < 0.05; **p < 0.01 ±

Table 1 List of primers used in this study

PCR Primer Sequence (5′-3′) Product Size (bp) PCR cycles Annealing temperature (°C)

RT-PCR ZNF471F GAG​ATG​ACG​AGT​GAG​ATG​AC 194 32 55 ZNF471R TGACTT​ CCC​ ATC​ TGC​ TTC​ TC​ GAPDHF GGA​GTC​AAC​GGA​TTT​GGT​ 206 23 55 GAPDHR GTG​ATG​GGA​TTT​CCA​TTG​AT qPCR ZNF471F GAG​ATG​ACG​AGT​GAG​ATG​AC 194 60 ZNF471R TGACTT​ CCC​ ATC​ TGC​ TTC​ TC​ GAPDHF CCA​GCA​AGA​GCA​CAA​GAG​GAA​ 114 60 GAPDHR GGT​CTA​CAT​GGC​AAC​TCA​AGG​ MSP ZNF471m44 TTTTGT​ TTT​ CGT​ TTT​ TTT​ CGTTC​ ​ 223 60 ZNF471m6 ACG​CGA​CTA​AAC​CTT​CGC​G ZNF471u44 GTTTTG​ TTT​ TTG​ TTT​ TTT​ TTG​ TTT​ ​ 228 58 ZNF471u6 AAA​AAC​ACA​ACT​AAA​CCT​TCACA​ BGS ZNF471BGS1 GGTTTT​ TTT​ TAT​ TTT​ TTT​ AGT​ AGT​ T​ 410 41 60 ZNF471BGS4 TCC​CAC​AAC​TCA​CTC​CAA​TAA​

Wound healing, migration, and Matrigel invasion assays confuent. Ten, the cell monolayer was scratched with a Stably-expressing ZNF471 and empty vector cells were sterile pipette tip, and the cells were washed once with plated in 6-well plates and cultured overnight until PBS and cultured in serum-free medium. Cell migration Tao et al. Clin Epigenet (2020) 12:173 Page 11 of 14

was observed under a phase-contrast microscope (Leica 10 nM siRNA. Te cells were collected for RNA or pro- DMI4000B, Milton Keynes, Bucks, UK) every 24 h. All tein extraction at 48–72 h after transfection. experiments were performed in triplicate. Transwell migration and invasion assays were per- In vivo tumorigenicity formed to assess cell migration and invasion. Transwell To verify the inhibitory efect of ZNF471 on tumors chambers (8-µm pore size; Corning, Inc., Corning, NY, in vivo, a nude mice tumorigenicity assay was per- USA) were used. Te cells were treated with serum star- formed. Female BALB/c nude mice (aged 4–6 weeks) vation overnight in the Transwell migration and invasion were obtained from the Experimental Animal Center of 4 assays. Cells were plated in the upper chamber at 2 × 10 Chongqing Medical University (CQMU; China). Stable cells/chamber in 200 μL serum-free medium, with 700 μL ZNF471-expressing MDA-MB-231 cells or empty con- 6 medium containing 10% FBS in the lower chamber. A trol cells (2.5 × 10 cells in 0.2 mL PBS) were injected into Matrigel (BD Biosciences) barrier was added on top of the left and right backs of nude mice. 21 days after injec- the Transwell membrane to verify cell invasiveness. After tion, tumors were derived from the mice and its size and incubation for 24 h, cells were fxed with 4% paraform- average weight were analyzed. Tumors were then fxed, aldehyde for 30 min and stained with crystal violet for dehydrated, and embedded in parafn for immunohis- 20 min. Cells were counted using microscopy. All experi- tochemistry and hematoxylin and eosin staining. Animal ments were performed in triplicate. experiments were approved by the ethics committee of CQMU. Western blot Cellular proteins were extracted and western blotting was Immunohistochemistry staining performed as previously described [26, 29, 30]. Protein Immunohistochemistry was performed as described pre- lysates (40 μg) were separated by sodium dodecyl sul- viously [17, 31]. Briefy, the tumor sections of nude mice fate‐polyacrylamide gel electrophoresis and transferred were dewaxed, rinsed, rehydrated, and antigen repaired. onto polyvinylidene fuoride membranes. Blocking was Te sections were then incubated overnight at 4 °C with performed using 5% milk for 1 h at room temperature. anti-Ki67 (1:400, ab15580; Abcam) antibody, followed Te following primary antibodies were used: ZNF471 by secondary antibody at 37 °C for 30 min. Diamin- (ab204974; Abcam), total AKT (#4691; Cell Signaling obenzidine (DAB) was used for color development and Technology, Danvers, MA, USA), phospho‐AKT (#13038; hematoxylin was used for counterstaining. Images were Cell Signaling Technology), total β-catenin (#9562; Cell captured under a microscope at 400 × magnifcation. Signaling Technology), active β-catenin (#4272; Cell Signaling Technology), p-GSK-3β (sc-373800), c‐Myc Statistical analysis (#13987; Cell signaling Technology), cyclin D1 (sc-450; For statistical analysis, Graphprism 7.0 (La Jolla, Califor- Santa Cruz Biotechnology), vimentin (sc‐6260; Santa nia) and SPSS23.0 software (Chicago, IL) were used. All Cruz Biotechnology), E‐cadherin (sc‐8426; Santa Cruz experimental analysis was performed in triplicate, and Biotechnology), N-cadherin (#14215; Cell Signaling results are expressed as the mean ± SD compared by Stu- Technology), Snail (#3879; Cell Signaling Technology), dent’s t-test. Univariate and multivariate Cox regression Slug (sc-166476; Santa Cruz Biotechnology), cleaved analyses were used for assessing the prognostic signif- caspase 9 (WL01838; Wanleibio, Shenyang, China), Bcl2 cance of ZNF471. p values < 0.05 demonstrated a signif- (#2870; Cell Signaling Technology), Bax (#502; Cell Sign- cant diference. aling Technology), cleaved-PARP (#7851; Cell Signaling Technology), p53 (sc-47698, Santa Cruz Biotechnology), Discussion MMP1/8 (sc-137044, Santa Cruz Biotechnology), and ZNF471 has been reported to be downregulated or MMP3 (sc-21732; Santa Cruz Biotechnology). Pro- silenced by promoter hypermethylation in gastric cancer, teins were imaged using an enhanced chemilumines- squamous cell carcinoma of the tongue and esophageal, cence detection system (Pierce Chemical Co., Rockford, and low expression of ZNF471 is associated with poor IL, USA). GAPDH was used as the protein expression survival [24, 25, 32]. However, its functions and mecha- control. nisms in breast cancer are unclear. In the present study, we found that ZNF471 was downregulated in six of nine Small interfering RNA (siRNA) breast cancer cell lines and 12 of 14 pairs of breast can- ZNF471 siRNA kits were purchased from OriGene ( cer tissues. Pharmacological demethylation experiments OriGene Technologies, Rockville, MD). Transfections and MSP demonstrated that its inactivation was due to were performed using Lipofectamine 2000 according to promoter CpG island methylation. Tese data suggested the manufacturer’s instructions with a concentration of ZNF471 could be a TSG in breast cancer. Tao et al. Clin Epigenet (2020) 12:173 Page 12 of 14

We thus investigated the functions of ZNF471 in breast squamous cell carcinoma, colorectal cancer, and breast cancer. Previous studies have shown that KRAB-ZFPs are cancer [21, 33, 43]. Whether or not ZNF471 could associated with cancer cell proliferation and are impor- modulate AKT and Wnt/β-catenin signaling in breast tant for other biological functions, including apoptosis cancer was still unclear. We found that overexpress- and migration/invasion in several tumors [21, 23, 33]. ing ZNF471 downregulated phospho-AKT, phospho- Terefore, the tumor-suppressive functions of ZNF471 GSK-3β, active-β-catenin, and their downstream target in breast cancer were frst investigated in this study. Te genes (c-Myc, cyclin D1); no signifcant changes were results demonstrated that ectopic expression of ZNF471 found in total AKT and β-catenin in MDA-MB-231 and in MDA-MB-231 and YCC-B1 cells signifcantly inhibited YCC-B1 cells. While phospho-AKT, phospho-GSK-3β, breast cell proliferation and colony formation, arrested active-β-catenin, c-Myc and cyclin D1 were all regu- the cell cycle at S phase, and promoted apoptosis. Analy- lated after knockdown of ZNF471. Tus, ZNF471 could sis of the expression of apoptotic markers confrmed its antagonize AKT and Wnt/β-catenin signaling. pro-apoptotic efect: cleaved caspase 9, cleaved-PARP, Bax, and p53 were upregulated while Bcl-2 was down- regulated in cells overexpressing ZNF471. In contrast, Conclusions knockdown of ZNF471 in BT549 cells promoted cell In summary, ZNF471 as a TSG in breast cancer and growth and inhibit apoptosis. In vivo, ZNF471 also inhib- often inactivated by promoter methylation. ZNF471 ited the formation of tumor xenografts in nude mice. inhibited cell growth, induced apoptosis, and arrested A signifcant decrease in cell migration and invasion the cell cycle; it also suppressed cell migration and ability was also observed after ZNF471 transfection. invasion. ZNF471 exerted its tumor suppressor func- We found that the highly aggressive cell morphology tion by reducing the EMT, inhibiting cell stemness, and was replaced by a less invasive morphology in ZNF471- blocking AKT and Wnt/β-catenin signaling. overexpressing cells. Te expression of the EMT markers Vimentin, Snail, Slug, and N-cadherin was decreased and Supplementary information the expression of E-cadherin was increased in ZNF471- Supplementary information accompanies this paper at https​://doi. overexpressing cells. While knockdown of ZNF471 org/10.1186/s1314​8-020-00959​-6. upregulated the expression of Vimentin, Snail, Slug, and N-cadherin. Tus, part of the mechanism of ZNF471 Additional fle 1: Table S1. Univariate and multivariate Cox regression analyses of ZNF471 in BC patients. inhibition of breast cancer cell metastasis was attenuat- Additional fle 2: Fig. S1. ZNF471 promoter methylation levels in breast ing the EMT. MMPs regulate growth, apoptosis, angio- tumor samples were detected by MSP. U: unmethylated. genesis, and cell stemness and are associated with tumor prognosis [34–37]. Increasing evidence has indicated that Abbreviations MMPs play a critical role in tumor metastasis by degrad- ZNF471: Zinc-fnger protein 471; KRAB-ZFP: Krüppel-associated box domain ing the extracellular matrix, and inducing and maintain- zinc fnger protein; RT-PCR: Semiquantitative RT-PCR; qPCR: Quantitative ing the EMT [38–40]. Te present study indicated that real-time PCR; CCK-8: Cell Counting Kit-8; MSP: Methylation-specifc PCR; BGS: Bisulfte genomic sequencing; TSG: Tumor suppressor gene; Aza: 5-Aza- ectopic expression of ZNF471 downregulated the expres- 2′-deoxycytidine; TSA: Trichostatin A; IBC: Invasive Breast Carcinoma; IDBC: sion of MMP1 and MMP3, while MMP1 was upregulated Invasive Ductal Breast Carcinoma; ILBC: Invasive Lobular Breast Carcinoma; after knocking down ZNF471. Taken together, ZNF471 EMT: Epithelial-mesenchymal transition; PBS: Phosphate-bufered saline; PI: Propidium iodide; DAB: Diaminobenzidine. suppressed breast cancer cell metastasis by attenuating the EMT and modulating MMPs. We next checked the Acknowledgements stem cell markers ABCG2, BMI1, KLF4, MAD2, STAT3, We thank Prof Sun Young Rha (Department of Internal Medicine, Yonsei Uni- versity, Korea) for some breast cell lines. CD44, MYC, NANOG2, and OCT4, the results of these experiments showed that ZNF471 could inhibit cell Authors’ contributions stemness in breast tumors. CT, LL, QT, XTX: conceptional design. CT, JL, JT, DZ, LL: performed experiments and analyzed data; ZQ, TCP: collected samples. CT, LL: prepared fgures and Te molecular mechanisms underlying the tumor- drafted the manuscript; LL, GR, QT: fnalized the manuscript. All authors read suppressive functions of ZNF471 in breast cancer were and approved the fnal manuscript. further analyzed. AKT and Wnt/β-catenin pathways Funding have been reported to participate in the development The study was supported by the National Natural Science Foundation of China of breast cancer, especially in cell proliferation and (NSFC) (#31420103915), Natural Science Foundation of Chongqing, China (No. metastasis [6, 41, 42]. KRAB-ZFPs have been shown cstc2020jcyj-bshX0025) and Hong Kong-RGC (GRF# 14115019). to suppress the growth of many tumors through Wnt/ Availability of data and materials β-catenin and AKT signaling, including esophageal The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Tao et al. Clin Epigenet (2020) 12:173 Page 13 of 14

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