ZNF143-Mediated H3K9 Trimethylation Upregulates CDC6 by Activating MDIG in Hepatocellular Carcinoma

Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/0008-5472.CAN-19-3226 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ZNF143-mediated H3K9 trimethylation upregulates CDC6 by activating MDIG in hepatocellular carcinoma

Lili Zhang1*, Qi Huo2*, Chao Ge1, Fangyu Zhao1, Qingqing Zhou1, Xiaoxia Chen3,

Hua Tian1, Taoyang Chen4, Haiyang Xie5, Ying Cui6, Ming Yao1, Hong Li1#, Jinjun

Li1#

1 State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute,

Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200032,

China

2 Department of Medical Oncology, The First Affiliated Hospital, School of Medicine,

Zhejiang University, Hangzhou 310003, China.

3 Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, China

4 Qi Dong Liver Cancer Institute, Qi Dong 226200, China

5 Department of General Surgery, the First Affiliated Hospital, School of Medicine,

Zhejiang University, Hangzhou 310000, China

6 Cancer Institute of Guangxi, Nanning 530027, China

* These authors contributed equally to this work.

# Co-corresponding authors

Running Title: ZNF143 promotes cell proliferation in HCC

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/0008-5472.CAN-19-3226 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

#Address for correspondence:

Jinjun Li, Ph.D. or Hong Li, Ph.D.

State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute,

Renji Hospital, Shanghai Jiaotong University School of Medicine

Add: 25/Ln 2200 Xietu Road, Shanghai 200032, China

E-mail: [email protected] or [email protected]

Tel: +86-21-64432140 or +86-21-64067346

Conflict of interest statement:

No potential conflicts of interest were disclosed.

Abstract

Zinc finger protein 143 (ZNF143) belongs to the zinc finger protein family and

possesses transcription factor activity by binding sequence-specific DNA. The exact

biological role of ZNF143 in hepatocellular carcinoma (HCC) has not been

investigated. Here we report that ZNF143 is overexpressed in HCC tissues and its

overexpression correlates with poor prognosis. Gain- and loss-of-function

experiments showed that ZNF143 promoted HCC cell proliferation, colony formation,

and tumor growth in vitro and in vivo. ZNF143 accelerated HCC cell-cycle

progression by activating cell division cycle 6 (CDC6). Mechanistically, ZNF143

promoted expression of CDC6 by directly activating transcription of histone

demethylase mineral dust-induced gene (MDIG), which in turn reduced H3K9me3

enrichment in the CDC6 promoter region. Consistently, ZNF143 expression

correlated significantly with MDIG and CDC6 expression in HCC. Collectively, we

propose a model for a ZNF143-MDIG-CDC6 oncoprotein axis that provides novel

insight into ZNF143 which may serve as a therapeutic target in HCC.

Introduction

Hepatocellular carcinoma (HCC) is the most common cause of death worldwide, and

the molecular mechanism of uncontrolled HCC progression remains unclear (1).

Accumulating evidence has suggested that dysregulation of the cell cycle is one of the

most important hallmarks of HCC (2). A selective CDK4/6 inhibitor showed

encouraging results in preclinical models of HCC and might represent a novel

therapeutic strategy for HCC treatment (3). Elucidation of the mechanisms underlying

the pathogenesis and molecular biology of dysregulated cell cycle in HCC is

fundamental for the development of effective therapeutic treatments.

Zinc finger protein 143 (ZNF143) can be activated by IGF1 and is a transcription

factor of the C2H2 family. This protein contains a transcription activation domain, 7

Kruppel-like C2H2 zinc finger motif domain, and a C-terminal domain (4,5). Some

studies have shown that ZNF143 possesses potent oncogenic properties and can

promote growth (6,7). In prostate cancer, ZNF143 is involved in cisplatin resistance

by regulating the transcription of DNA repair genes (8). Additionally, ZNF143 is

closely related to tumor malignancy via regulating cell motility and exerting tumor

suppressive effects in breast cancer (9). Intriguingly, Haibara et al. found that two

small molecules inhibit ZNF143 activity, suggesting an opportunity for cancer

therapeutics (10). To date, the exact biological role of ZNF143 in HCC has not been

investigated.

An increasing body of evidence suggests that epigenetic changes such as DNA

methylation and other post-translational modifications of chromatin play critical roles

in HCC tumorigenesis (11,12). Of these epigenetic alterations, dynamic histone

modifications play a pivotal role in the regulation of gene transcription (13). Histone

methylation can be regulated by histone methyltransferase and demethylases. Mineral

dust-induced gene (MDIG), also known as myc-induced nuclear antigen with a

molecular weight of 53 kDa (MINA53), was first identified in alveolar macrophages

obtained from coal miners (14), contains one JmjC domain and can demethylate

H3K9me3 (15,16). Numerous studies have demonstrated that MDIG is overexpressed

in malignancies and can promote cell migration, cell cycle transition and proliferation

(14,17).

Various studies have shown that functionally related genes involved in epigenetic

reprogramming can be controlled by specific transcription factors (18,19). Elucidation

of the epigenetic alterations induced by transcription factors will provide more

comprehensive and precise insight into the regulatory mechanisms of transcription

factors. In the current study, we identified cell division cycle 6 (CDC6) as a

downstream target gene of ZNF143 during the regulation of cell cycle in HCC.

ZNF143 promoted CDC6 expression by attenuating the enrichment of H3K9me3 in

CDC6 promoter region through activation of MDIG. Our findings reveal the

fundamental roles of ZNF143 in cell cycle control and identify the

ZNF143-MDIG-CDC6 axis as a potential target for anticancer therapy.

Materials and Methods

Human tissues

Human primary HCC/matched adjacent noncancerous liver tissue specimens were

obtained with informed consent from the Guangxi Cancer Institute (Nanning, China),

Zhejiang University (Hangzhou, China) and the Qidong Liver Cancer Institute

(Qidong, China). None of the patients received preoperative radiation or

chemotherapy. This study was approved by the Research Ethics Committee of Renji

Hospital, Shanghai Jiao Tong University School of Medicine and in accordance with

Declaration of Helsinki. Written informed consent was received from participants

prior to inclusion in the study.

The Cancer Genome Atlas (TCGA) and HCCDB cohort

The mRNA expression data were downloaded from TCGA database

(https://tcga-data.nci.nih.gov/tcga/). HCCDB data were directly downloaded from the

HCCDB dataset (http://lifeome.net/database/hccdb/home.html) (20).

Immunohistochemistry (IHC)

Tissue microarrays (TMAs) were constructed, and the diagnosis was confirmed by

three pathologists. IHC was performed as previously described (21). Briefly, the

sections were incubated overnight with ZNF143, MDIG and CDC6 at 4°C and then

evaluated by two independent observers in a blinded manner. The antibodies used in

this study were anti-ZNF143 (HPA003263, 1:10, Sigma-Aldrich),

anti-MDIG/MINA53 (PA5-31300, 1:25, Invitrogen), anti-CDC6 (HPA050114, 1:10,

Sigma-Aldrich) and anti-Ki67 (GT209429, 1:50, GeneTech). Scores of staining

intensity were: 0, negative; 1, weak; 2, moderate; 3, strong. Scores of positively

stained cell proportion were: 0, no positive; 1, <10%; 2, 10%–35%; 3, 35%–75%;

4, >75%.The results were scored 0 to 4 by two independent investigators. A score of

0–2 was considered to represent low expression and a score of 3–4 was considered to

represent high expression.

Cell culture

The human HCC cell lines MHCC-97L, MHCC-LM3, and MHCC-97H were kindly

provided by the Liver Cancer Institute of Zhongshan Hospital, Fudan University

(Shanghai, China). Huh7 was obtained from the Riken Cell Bank (Tokyo, Japan). Li-7

cells were purchased from the Cell Bank of the Institute of Biochemistry and Cell

Biology, China Academy of Sciences (Shanghai, China). The Hep 3B and HEK-293T

cell lines were obtained from the American Type Culture Collection (ATCC)

(Manassas, VA, USA). Cells were all cultured in Dulbecco’s modified Eagle’s

medium (DMEM; Sigma-Aldrich) containing 10% fetal bovine serum (FBS, Gibco,

New York, USA) and maintained at 37°C with 5% CO2. All cell lines were

authenticated by Short Tandem Repeat (STR) profiles in the past six months and were

used within 10 passages after reviving from the frozen stocks. Cells were free of

Mycoplasma contamination was determined by PCR assay.

Plasmid constructs, lentivirus and siRNA

The human ZNF143 cDNA (NM_003442.6) and CDC6 cDNA (NM_001254.4) was

subcloned into the GV492 vector (Genechem, Shanghai, China). ShRNAs targeting

ZNF143 (shZNF143#1/shZNF143#2/shZNF143#3), CDC6 (shCDC6#1/shCDC6#2)

and their negative control (shNC) were obtained from Genechem (Shanghai, China).

Target sequences are listed in Supplementary Table 1. The cDNA sequences of human

MDIG and shMDIG target sequences were described in our previous study (21).

The process of lentivirus production and cell transfection was performed as described

previously (21). SiRNAs targeting CBX3 (siCBX3#1/siCBX3#2) and CBX5

(siCBX5#1/siCBX5#2) and their negative control (siNC) were obtained from

GenePharma (Shanghai, China). Target sequences are listed in Supplementary Table

RNA isolation and qPCR assay

Total RNA was isolated with TRIzol reagent (Invitrogen, CA) and reverse-transcribed

into cDNA by using the PrimeScriptTM RT Reagent Kit (RR037A, TaKaRa Bio,

Japan). Quantitative real-time polymerase chain reaction assays were carried out by

using TB GreenTM Premix Ex TaqTM II (RR820A, TaKaRa Bio, Japan) on an Applied

Biosystems 7500 Software version 2.0.5 real-time PCR system (Thermo Scientific,

USA) according to the manufacturer’s instructions. GAPDH was used as an internal

loading control. The primer sequences are listed in Supplementary Table 2.

Western blot analysis.

Western blot analysis was performed as previously described (22). Antibodies used in

this study were anti-ZNF143 (sc-100983, 1:200, Santa Cruz), anti-MDIG (ab155335,

1:200, Abcam), anti-CDC6 (sc-9964, 1:50, Santa Cruz), anti-H3K9me3 (ab8898, 1:50,

Abcam), anti-β-Actin (A1978, 1:10000, Sigma-Aldrich), anti-Rabbit IgG (A0545,

1:5000, Sigma-Aldrich), and anti-Mouse IgG (A4416, 1:5000, Sigma-Aldrich),

anti-MDIG (39-7300, 1:200, ThermoFisher), anti-CBX3(11650-2-AP, 1:200,

Proteintech), anti-CBX5(11831-1-AP, 1:200, Proteintech), anti-Flag (F1804, 1:200,

Sigma-Aldrich).

Transient transfection

The transfections of the plasmid DNAs and siRNAs were carried out with

Lipofectamine 2000 transfection reagent (Invitrogen) following the manufacturer’s

instructions.

MTT assay

A total of 1000 cells were seeded in 96-well plates in 3 replicates. Next, 10 µl MTT

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent (5 mg/ml,

Sigma-Aldrich) was added to each well and incubated for 4 h at 37°C. Then, the

media were removed, and 100 μl DMSO (Sigma-Aldrich) was added to the well. The

OD value was measured at 570 nm every 24 h.

Colony formation assay

Colony formation assay was performed according to previous methods (21). A total of

1000 cells were seeded into 6-well culture plates and cultured for approximately 15

days. Then, the cells were washed twice with PBS, fixed in neutral buffered formalin

for 30 min, and stained with Giemsa (Sigma-Aldrich) for another 30 min. Three

independent experiments were performed.

Flow cytometry analysis

Flow cytometry analysis was performed as previously described (22). In brief, 1 × 106

cells were plated into 6-well plates. After adhering to the well, the cells were treated

with 2 mM thymidine (Sigma-Aldrich) for 24 h and harvested by trypsin after

releasing for 0 and 24 h. Then, the cells were washed with PBS twice and fixed with

70% ethanol at -20°C overnight. Before analysis by flow cytometry, the cells were

washed twice with 1x PBS and resuspended in 200 μl PI solution [50 μg/ml PI

(Sigma-Aldrich), 100 μg/ml RNase (Sigma-Aldrich), 0.2% Triton X-100] at 4°C for

30 min. The results were analyzed by Modfit 3.2 software. Three independent

experiments were performed.

In vivo tumor formation assay

For the tumor xenograft assays, 2 × 106 Li-7 cells infected with ZNF143/Vector, 2 ×

106 MHCC-97H cells transfected with shNC/shZNF143#2/shZNF143#3, 2 × 106 Li-7

cells infected with Vector + shNC/ZNF143 + shNC/ZNF143 + shMDIG#2/ ZNF143 +

shMDIG#3, or 2×106 MHCC-97H cells transfected with shNC/ shCDC6#1

/shCDC6#2 were resuspended in 200 μl of serum-free DMEM and inoculated

subcutaneously into one flank of each nude mouse (nu/nu, male, 4 weeks, n = 9 per

group). After 4 weeks, all mice were sacrificed, and the tumor weights were measured.

The animal experimental protocols were approved by the Shanghai Medical

Experimental Animal Care Commission and performed in accordance with the

institutional ethical guidelines for animal experiments.

Dual luciferase assay

A dual luciferase assay was performed as previously described (21). The normal and

mutant promoter region of MDIG was subcloned into the pGL3 vector (Promega,

Madison, WI). The primers for cloning are provided in Supplementary Table 3 (21,23).

MHCC-97L and MHCC-LM3 cells were transiently transfected with the

corresponding pGL3 reporter constructs and the PRL-TK reporter plasmid together

with ZNF143 or Vector using Lipofectamine 2000 (Invitrogen). Cells were lysed at 48

h after transfection. The dual luciferase assay reporter system was used according to

the manufacturer’s instructions (Promega).

Chromatin immunoprecipitation (ChIP) assay

MHCC-97H/Hep 3B/Huh7 cells, and MHCC-97H/ Huh7 cells transfected with

shNC/shMDIG#3 or siNC/siCBX3#2/siCBX5#1, and Li-7/MHCC-LM3 cells

overexpressing ZNF143/Vector, and MHCC-97H/Hep 3B cells transfected with

shNC/shZNF143#2 were fixed and immunoprecipitated using the EZ-Magna ChIP

assay kit as recommended by the manufacturer (Millipore, Billerica, MD, USA).

Antibodies used to immunoprecipitate purified chromatin were anti-H3K4me3

(ab213224, Abcam), anti-H3K9me3 (ab8898, Abcam), anti-H3K27me3 (ab6002,

Abcam), and anti-ZNF143 (16618-1-AP, Proteintech), anti-MDIG (39-7300,

ThermoFisher), anti-CBX3 (11650-2-AP, Proteintech), anti-CBX5 (11831-1-AP,

Proteintech). Primers used to amplify the promoter regions of MDIG, CDC6 or other

S phase genes are shown in Supplementary Table 2.

Co-Immunoprecipitation (Co-IP) assay

Co-IP assays were performed using MHCC-97H/Huh7 cells, and MHCC-97L/

MHCC-LM3 cells overexpressing ZNF143. The cells were harvested in RIPA

(Upstate, Biotechnology) lysis buffer for 40 min on ice and centrifuged at 12 000 g

for 10 min. The protein A/G agarose beads were incubated with antibody overnight at

4 °C while rotating. After washing, the complexes were subjected to western blotting

analysis. Antibodies used were anti-MDIG (12214-1-AP, Proteintech), anti-CBX3

(11650-2-AP, Proteintech), anti-CBX5 (11831-1-AP, Proteintech), anti-Flag (F1804,

Sigma-Aldrich).

mRNA profiling and bioinformatics analysis

For mRNA profiling analysis, MHCC-97H and Huh7 cells were stably transfected

with shMDIG#3 or shNC and then subjected to a human gene expression microarray

(Affymetrix) assay (GeneTech, Shanghai, China). KEGG pathway analysis (genes

with P < 0.05, fold change > 1.5) was carried out by DAVID software

(https://david.ncifcrf.gov).

Statistics

Statistical analysis was carried out using SPSS 19.0 software and GraphPad Prism 5

and Image J software. Two-tailed and unpaired Student’s t-tests were used for two

group comparisons. The differences in ZNF143, MDIG, and CDC6 expression levels

between the paired HCC tissues and adjacent nontumorous liver tissues were

compared by paired t-tests. Pearson correlation analysis was performed to analyze the

correlation of two molecules. Survival curves were estimated using the Kaplan-Meier

method and compared using the log-rank test. Data are shown as the mean ± SD. P <

0.05 was considered statistically significant.

Results

ZNF143 is overexpressed and associated with poor prognosis in HCC

To investigate the role of ZNF143 in HCC, we first analyzed ZNF143 mRNA

expression in TCGA database. As shown in Fig. 1A, ZNF143 expression in HCC

samples was significantly higher than that in their matched benign counterpart. We

then examined the mRNA and protein expression of ZNF143 in HCC and matched

noncancerous tissues from our lab by qPCR and Western blot analyses. Consistent

with the results of TCGA dataset, ZNF143 was upregulated in HCC tissue compared

to control tissue (Fig. 1B-D). The HCCDB dataset showed that HCC is one of the

tumors displays obvious upregulation of ZNF143 (Supplementary Fig. 1A) (20).

These results indicated that ZNF143 expression is substantially increased in HCC.

Notably, patients with ZNF143 overexpression exhibited worse overall survival in

TCGA dataset (Fig. 1E). Moreover, ZNF143 expression was positively correlated with

histological grades in TCGA dataset (Fig. 1F). These results suggested that

upregulation of ZNF143 contribute to poor clinical outcomes of HCC patients.

ZNF143 promotes HCC cell growth

To evaluate the biological effects of ZNF143 in HCC, we first examined ZNF143

expression in immortalized normal hepatocyte L-02 cell and a series of HCC cell lines

by Western blot analyses (Supplementary Fig. 2A). We stably established

ZNF143-overexpressing cell lines in MHCC-97L, MHCC-LM3, Li-7 cells

(Flag-tagged ZNF143/Vector) and ZNF143 knockdown cell lines in Hep 3B,

MHCC-97H cells (Mock/shNC/shZNF143#1/shZNF143#2/shZNF143#3) with

lentiviral transfection. The protein level of ZNF143 in ZNF143-overexpressing or

knockdown HCC cells were verified by Western blot (Supplementary Fig. 2B). MTT

assays indicated that ectopic expression of ZNF143 promoted HCC cell proliferation,

whereas knockdown of ZNF143 led to a marked reduction in proliferation (Fig. 2A

and B). Plate colony formation assays demonstrated that ZNF143 enhanced the

clonogenicity of HCC cells (Fig. 2C and D). To extend the in vitro results, we

explored the role of ZNF143 in tumor growth using a xenograft model. As shown in

Fig. 2E and F, in nude mice, when ZNF143 was overexpressed in Li-7 cells, the

tumors showed enhanced growth. In contrast, tumors with shZNF143#2 and

shZNF143#3 exhibited smaller volumes and lower weights than tumors with shNC in

MHCC-97H cells. Western blot showed that ZNF143 expression remained high in

xenografts from ZNF143-overexpressing cells and low in xenografts from ZNF143

knockdown cells (Supplementary Fig. 2C). Moreover, the Ki67 percentage score of

tumor cells was relatively increased in ZNF143-overexpressing group and decreased

in shZNF143 group when compared with their negative control (Fig. 2G). The

expression of ZNF143 was positively correlated with Ki67 and PCNA in TCGA

dataset (Fig. 2H). Altogether, these findings suggested that ZNF143 is a positive

regulator of HCC proliferation.

ZNF143 promotes HCC cell cycle progression by enhancing CDC6 expression

To further clarify the effect of ZNF143 on HCC cell proliferation, we evaluated the

cell cycle distribution by flow cytometry after upregulating and knocking down

ZNF143 expression in HCC cell lines. As shown in Supplementary Fig. 2 D and E,

ZNF143 knockdown in MHCC-97H cells led to G1/S arrest, together with a

substantial decline in the cell population in S phase, and ZNF143 overexpression

significantly facilitated the cell-cycle transition by increasing the population of S

phase cells and decreasing the population of G1 phase cells among MHCC-LM3 cells.

Then, we further added 2 mM thymidine to synchronize cells at the G1/S phase border.

After release for 24 h, flow cytometry analysis showed that the percentage of cells at

G1 phase was significantly lower in ZNF143-overexpressing MHCC-LM3 cells and

was higher in ZNF143 knockdown MHCC-97H cells than their corresponding control

cells (Fig. 3A and B). These results suggested that ZNF143 is crucial for HCC

cell-cycle progression in the G1/S transition.

We next explored the mechanism by which ZNF143 regulates cell proliferation and

cell cycle progression. Given the role of ZNF143 in the G1/S transition, we examined

the mRNA expression of S phase genes in HCC cells after ZNF143 overexpression

and knockdown by qPCR analyses (Fig. 3C) (24). Among these genes, CDC6

attracted our attention because of its marked change, and its regulatory mechanism in

HCC has not been fully elucidated (25,26). Western blot results further showed that

ZNF143 could activate CDC6 at the protein level (Fig. 3D). Then, we evaluated

biological effects of CDC6 in HCC cells. We established stable overexpression of

CDC6 (CDC6/Vector) in MHCC-97L and MHCC-LM3 cells and stable knockdown

of CDC6 (Mock/shNC/shCDC6#1/shCDC6#2) in MHCC-97H and Huh7 cells. The

protein expression levels of CDC6 in HCC cells were verified by Western blot

(Supplementary Fig. 3A). The plate colony formation and MTT assays revealed that

CDC6 enhanced HCC cells proliferation and clonogenicity (Supplementary Fig.

3B-E). In xenograft model, the tumors from MHCC-97H-shCDC6 cells showed less

active proliferative ability at the implantation site than control (Supplementary Fig. 3F

and G). Flow cytometry further suggested that CDC6 promotes G1/S transition in

HCC cells (Supplementary Fig. 3H and I). In addition, the plate colony formation and

MTT assays revealed that CDC6 knockdown inhibited cell proliferation induced by

ZNF143 overexpression in HCC cells, and CDC6 re-expression in the

ZNF143-knockdown cells rescued the proliferation ability (Fig. 3E-J and

Supplementary Fig. 4A-E).

ZNF143 enhancing CDC6 expression via the alternation of H3K9me3

enrichment on CDC6 promoter

Global changes in the epigenetic landscape are a hallmark of cancer (27). A set of

functionally related genes involved in epigenetic reprogramming can be controlled by

specific transcription factors (28-30). Here, we investigated whether ZNF143

modulates epigenetic changes during activation of CDC6. In contrast to other histone

modifications, histone methylation has been highlighted because of its highly specific

dynamics with respect to gene regulation (31). Here, we focused on the distribution of

the active marker H3K4me3 and the repressive markers H3K27me3 and H3K9me3 on

the promoter region of CDC6. Six pairs of primers (Supplementary Table 2) were

used to detect possibly altered sites in CDC6 promoter region (Supplementary Fig.

4F). As shown in Fig. 4A and B, ChIP-qPCR assays indicated that the enrichment of

H3K9me3 was obviously decreased at -0.2 kb and +0.2 kb promoter regions of CDC6

after ZNF143 overexpression in both MHCC-LM3 and Li-7 cells, and ZNF143

knockdown increased H3K9me3 at CDC6 promoter in MHCC-97H and Hep 3B cells.

The enrichment of H3K4me3 and H3K27me3 on CDC6 promoter showed different

results in ZNF143-overexpressing HCC cells (Supplementary Fig. 4G-J). Taken

together, our data suggest that alterations in histone methylation, especially H3K9me3,

might contribute to the activation of CDC6 by ZNF143.

We further examined the mechanism underlying ZNF143 regulates H3K9me3 in HCC.

Histone methylation is regulated by two classes of enzymes with opposing activities:

histone methyltransferases and histone lysine demethylases (31-33). Given role in the

regulation of cancer growth, we focused on histone lysine demethylases of

H3K9me3(31). Based on previous studies, we selected KDM4A, KDM4B, KDM4C,

KDM4D, and MDIG (MINA53) for further analysis (32,34). qPCR showed that

ZNF143 upregulated MDIG expression (Fig. 4C). MDIG is overexpressed in a variety

of human cancers and plays a key role in cell proliferation (16,21,35). We then

examined the protein expression of MDIG in HCC cells with altered ZNF143

expression. As shown in Fig. 4D, the MDIG protein level was upregulated in

ZNF143-overexpressing HCC cells and significantly inhibited in ZNF143 knockdown

HCC cells.

To further elucidate the mechanism by which ZNF143 activates MDIG expression in

HCC, we analyzed the MDIG promoter region for possible ZNF143 binding sites.

JASPAR software indicated that two potential ZNF143 DNA binding motifs exist in

the promoter region of MDIG (Fig. 4E and F). Luciferase assays showed that relative

luciferase activity of the MDIG promoter was significantly induced by ZNF143

overexpression, and mutation of binding site 1, but not other sites, abolished

ZNF143-mediated induction of the MDIG promoter reporter activity (Fig. 4G). ChIP

assay also indicated that endogenous ZNF143 was recruited to binding site 1 of the

MDIG promoter (Fig. 4H). These data suggested that ZNF143 promotes MDIG

transcription by binding to its promoter.

MDIG promotes cell cycle progression by enhancing CDC6 expression in HCC

A series of studies have shown that MDIG plays an important role in tumorigenesis

(14,16,36). Our previous study showed that MDIG overexpression promoted HCC

cell proliferation, migration and spreading (21). Gene expression profiling of

MDIG-knockdown (shMDIG) vs. negative control (shNC) revealed that 66 genes,

including CDC6, were downregulated and 32 genes were upregulated after MDIG

was knocked down in both MHCC-97H and Huh7 cells (Fig. 5A and Supplementary

Table 4). The enriched KEGG pathways of 66 overlapping genes included the cell

cycle (Fig. 5A). Therefore, we explored the role of MDIG in cell cycle progression.

The results showed that MDIG knockdown in Huh7 cells led to G1/S arrest.

Overexpression of MDIG in MHCC-LM3 cells contributed to G1/S phase transition

(Supplementary Fig. 5A-G). These results suggested that MDIG is crucial for HCC

cell cycle progression.

We next examined whether CDC6 expression can be regulated by MDIG. As shown

in Fig. 5B and Supplementary Fig. 5H and I, Western blot and qPCR analyses

indicated that MDIG promoted the expression of CDC6. Our previous study showed

that MDIG could suppress the formation of H3K9me3 (21). We hypothesized that

alterations of epigenetic modifications might contribute to the increase of CDC6, and

the enrichment of H3K9me3 in CDC6 promoters was analyzed. Six pairs of primers

were used to detect possibly altered sites in CDC6 promoter (Supplementary Fig. 4F).

As shown in Fig. 5C, the enrichment of H3K9me3 was specifically increased at the

promoter regions of CDC6 in MHCC-97H and Huh7 cells after MDIG knockdown. In

addition, H3K9me3 enrichment in the promoter region of genes regulating S phase

entry were analyzed by ChIP-qPCR. After knocking down MDIG, the enrichment of

H3K9me3 was increased at the promoter regions of CCNA2, MCM3, CDC6 in

MHCC-97H cells and CCNA2, MCM3, CDC6, TK1 in Huh7 cells (Fig. 5D). ChIP

assay also showed the enrichment of MDIG was found at the promoters of these genes

(Fig. 5E and Supplementary Fig. 5J). Then, we next try to explore the mechanism

mediating the recruitment MDIG to CDC6 promoter. ZNF143 is a transcription factor,

we first speculated whether ZNF143 mediates this recruitment.

Co-immunoprecipitation (co-IP) showed that endogenous MDIG and Flag-tagged

ZNF143 cannot interact with each other (Supplementary Fig. 6A and B). CBX3 and

CBX5 encoding HP1 proteins can recognize and bind to H3K9me3 (37). Previous

study showed a protein interaction between MDIG and CBX3, CBX5 (38). We next

determine whether CBX3 or CBX5 mediates this recruitment. Co-IP of endogenous

proteins confirmed the interaction between MDIG and CBX3 or CBX5

(Supplementary Fig. 6A, C and D). ChIP assay further demonstrated the enrichment

of CBX3, CBX5 on CDC6 promoter region (Supplementary Fig. 6E). We next

investigated the effect of HP1 proteins depletion on the recruitment of MDIG on

CDC6 promoter region. We silenced the expression of CBX3 or CBX5 by siRNA

(Supplementary Fig. 6F and G). After knocking down CBX3, MDIG was reduced at

CDC6 promoter region both in MHCC-97H and Huh7 cells. However, after knocking

down CBX5, the enrichment of MDIG at CDC6 promoter was decreased only in

MHCC-97H cells (Supplementary Fig. 6H). The mRNA expression of CDC6 was

decreased mainly after decreasing CBX3 (Supplementary Fig. 6I and J). The result

showed that CBX3 plays a major role in this recruitment in HCC cells. Interestingly,

ZNF143 knockdown also decreased the enrichment of CBX3 in CDC6 promoter

region (Supplementary Fig. 6K). In addition, MTT and Colony formation assays

showed that knocking down CDC6 inhibited the proliferation induced by MDIG

overexpression (Fig. 5F-H and Supplementary Fig. 6L).

ZNF143 promotes CDC6 expression by activating MDIG

To further examine whether ZNF143 promotes CDC6 expression by activating MDIG,

we first examined the expression of MDIG, CDC6 and H3K9me3 after ZNF143

overexpression and knockdown by Western blot analyses. As shown in Fig. 6A and B,

MDIG and CDC6 were upregulated and H3K9me3 was decreased in

ZNF143-overexpressing HCC cells. After ZNF143 knockdown, the opposite effects

were observed (Fig. 6C and D). In addition, similar results were observed in

ZNF143-overexpressing mouse tumor xenografts (Supplementary Fig. 6M). ZNF143

up-regulated the expression of MDIG and CDC6 in xenograft tumor tissue derived

from Li-7 cells with ZNF143 overexpression and MHCC-97H cells with ZNF143

knockdown were also demonstrated by immunohistochemistry analysis

(Supplementary Fig. 7A). To determine the role of MDIG in the upregulation of

CDC6 induced by ZNF143, we knocked down MDIG under conditions of ZNF143

overexpression. The results showed that the upregulation of CDC6 induced by

ZNF143 overexpression was inhibited and H3K9me3 was increased by MDIG

knockdown (Fig. 6E, F and Supplementary Fig. 7B and C). Meanwhile, the

enhancement of tumor growth, cell proliferation and colony formation induced by

ZNF143 overexpression were inhibited by MDIG knockdown in HCC cells (Fig. 6

G-I and Supplementary Fig. 7D-F). The protein levels of ZNF143 and MDIG in

mouse tumor tissues were examined by Western blot analyses (Supplementary Fig.

7G). In addition, the decreased proliferation and clonogenicity induced by shZNF143

could be rescued by MDIG overexpression in ZNF143-knockdown cells

(Supplementary Fig. 7H-K).

Clinical correlations among ZNF143, MDIG and CDC6

Given that ZNF143 increased CDC6 expression by upregulating MDIG, we first

analyzed the expression of MDIG and CDC6 in HCC and matched adjacent nontumor

liver tissues in TCGA dataset and our HCC patient tissues. As shown in

Supplementary Fig. 8A and B, the relative mRNA expression of MDIG and CDC6

was higher in most HCC tissues than their matched nontumorous liver tissues.

Kaplan–Meier survival analysis indicated that patients with high expression of MDIG

or CDC6 showed significantly worse overall survival in TCGA dataset

(Supplementary Fig. 8C). The mRNA level of ZNF143 was positively correlated with

MDIG and CDC6 expression in HCC tissues (Fig. 7A and B). The positive

correlations among ZNF143, MDIG and CDC6 at the protein level were also

demonstrated by immunohistochemistry analysis of 211 HCC patients (Fig. 7C, D and

Supplementary Fig. 8D). Intriguingly, the ZNF143 and MDIG protein abundance

fluctuated conformably during the cell cycle progression in HEK-293T cells

(Supplementary Fig. 8E). These data confirmed that ZNF143 expression was

correlated with MDIG and CDC6 expression and was clinically relevant in HCC.

Taken together, our data support a role for the ZNF143/MDIG/CDC6 axis in

promoting HCC progression and suggest that ZNF143 might serve as a biomarker and

potential target for HCC diagnosis and therapy (Fig. 7E).

Discussion

Dysregulation of the cell cycle leading to hyperactive cell division is often found in

cancers, and sustained proliferative signaling is a well-recognized hallmark of

malignancy (39,40). Pharmacologic inhibitors of cyclin-dependent kinases have

shown promising effects in patients with breast and other cancers (41). In this study,

our data showed that ZNF143 promotes HCC cell proliferation, colony formation,

tumor growth and cell cycle transition.

Epigenetic alterations associated with cell cycle disorders are common phenomena in

various cancers, including HCC (32,33). A series of studies have shown that

transcription factors work in concert with histone modification enzymes to exert their

function as gene regulators (18,42). ZNF143 is a novel chromatin-looping factor that

contributes to the architectural foundation of the genome by providing sequence

specificity at promoters (43). The CDC6 protein is essential for initiation of DNA

replication and is overexpressed in various cancers (25,44). In this study, we found

that CDC6 plays a role in cell proliferation of HCC. Intriguingly, our results showed

that ZNF143 regulates the expression of CDC6 through epigenetic alteration of

H3K9me3 modification by activating MDIG. MDIG is a cell growth regulating gene

that contains a JmjC domain and suppresses the formation of H3k9me3 (14,45). We

identified MDIG as a direct target of ZNF143. ZNF143 overexpression significantly

promoted MDIG expression and decreased H3K9me3 expression in HCC. Our

previous study showed that MDIG overexpression promoted HCC cell proliferation,

cell migration and invasion (21). In this study, we further demonstrated that MDIG

enhances HCC cell cycle progression. Our result showed that beyond CDC6, further

genes regulating S phase entry can be regulated by MDIG and H3K9me3, we believed

that MDIG-dependent histone demethylation of promoters of S phase genes might a

general mechanism to control gene expression in the S phase. In addition, we

demonstrated that mainly CBX3 mediate recruiting MDIG to CDC6 promoter.

Heterochromatin protein 1 (HP1) proteins interact with other molecules were

originally identified as critical components in heterochromatin mediated gene

silencing (37). Interestingly, our result showed HP1 proteins did not exert

transcriptional repression on CDC6 expression. After knocking down HP1 proteins

especially CBX3 slightly decreased CDC6 expression. In addition, whether other

molecular medicated the recruitment MDIG to the CDC6 promoter will be conducted

further research in the future. All these results further elucidate the role of ZNF143.

Our study also showed that ZNF143 is overexpressed in HCC tissue compared with

adjacent normal tissue. Furthermore, by analyzing the clinical features, we identified

ZNF143 as a potential biomarker of reduced survival in HCC. Paek et al.

demonstrated that IGF-1 induces the expression of ZNF143 in colon cancer cells

through phosphatidylinositide 3-kinase (PI3-kinase) and reactive oxygen species (4).

Whether wortmannin, an inhibitor of PI3-kinase, diphenyleneiodonium (DPI), an

NADPH oxidase inhibitor, and monodansylcardavarine (MDC), a receptor

internalization inhibitor, have therapeutic effects in HCC with high ZNF143

expression needs to be studied in the future. More importantly, Haibara et al.

discovered two novel small molecules that inhibit ZNF143 activity (10). All these

discoveries provided the opportunity that ZNF143 might be a therapeutic target

during HCC progression. In addition, future studies should also aim at characterizing

the protein complexes engaged by ZNF143 in order to provide other rational for novel

drugs that can inhibit the function of ZNF143 in HCC cells.

To the best of our knowledge, our report is the first attempt to validate the oncogenic

role of ZNF143 in HCC, our data suggested that ZNF143 is a prognostic biomarker

and is overexpressed in HCC tissues. ZNF143 promotes proliferation by activating

cell cycle progression in HCC cells. Importantly, we proposed a novel model for the

ZNF143-MDIG-CDC6 regulatory axis, which might provide insight into the function

of ZNF143 in HCC development.

Acknowledgments

This work was supported in part by grants from the National Key Program for Basic

Research of China (973) (2015CB553905), National Natural Science Foundation of

China (81972580, 81773152, 81672832), the National Key Sci-Tech Special Project

of China (2018ZX10723204-006), Key Discipline and Specialty Foundation of

Shanghai Municipal Commission of Health and Family Planning (2018BR20).

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Figure legends

Figure 1. ZNF143 is overexpressed and associated with poor prognosis in HCC.

A, ZNF143 expression in 49 cases of HCC and the corresponding nontumorous liver

tissues in The Cancer Genome Atlas (TCGA) dataset (log2TPM). (Left panel:

two-sided unpaired t-test, Right panel: two-sided paired t-test). B, ZNF143 mRNA

expression in 30 paired HCC tissues and the adjacent matched noncancerous tissues in

our laboratory were determined by qPCR. For qPCR, values were normalized with

GAPDH. (Left panel: two-sided unpaired t-test, Right panel: two-sided paired t-test).

C, The protein levels of ZNF143 in 24 paired HCC (T) and adjacent normal (N)

samples were measured by Western blot. β-Actin was used as a loading control. D,

Waterfall plot showing the protein level of ZNF143 in HCC compared with adjacent

noncancerous tissues from 24 patients determined by Western blot assays. (Red

histogram: ZNF143 overexpressed more than 2 times. Blue histogram: ZNF143

decreased more than 2 times). E, The correlation between ZNF143 expression and

overall survival of HCC patients in TCGA dataset (n=332) was assessed by Kaplan–

Meier survival analysis (Cut-off value: 3.15). F, ZNF143 expression in early-stage

(stage I + II, n=248) and advanced-stage (stage III + IV, n=87) HCC tissues in TCGA

dataset (log2TPM). The data were presented as mean ± SD. P values: *P < 0.05; **P

< 0.01; ***P < 0.001 by two-tailed Student t test (A, B and F), or by Kaplan–Meier

log rank test (E).

Figure 2. ZNF143 promotes HCC cell proliferation in vitro and in vivo. A and B,

The growth capacity of stably transfected ZNF143 overexpression (A) or knockdown

(B) cell lines was monitored with MTT assays. C and D, Representative images of

colony formation in ZNF143-overexpressing MHCC-97L/MHCC-LM3/Li-7 cell lines

quantitative analysis data. E and F, Subcutaneous tumor formation in nude mice (n =

9/group). Li-7 with ZNF143 overexpression (E) and MHCC-97H cells with ZNF143

knockdown (F) were injected into one flank of the mouse. Tumors were weighed. G,

Immunohistochemical images of Ki67 expression in xenograft tumors derived from

Li-7 cells with ZNF143 overexpression and MHCC-97H cells with ZNF143

knockdown. Original magnification: × 400. The positively stain (in percentages) were

analyzed (right panel) H, The correlation between ZNF143 and Ki67 or PCNA

mRNA expression in TCGA dataset (log2TPM). Data represent the mean ± SD of at

least three independent replicates. P values: *P < 0.05; **P < 0.01; ***P < 0.001 by

two-tailed Student t test (A-G), or by pearson correlation analysis (H).

Figure 3. ZNF143 promotes HCC cell cycle progression by enhancing CDC6

expression. A and B, MHCC-LM3 with ZNF143 overexpression (ZNF143/Vector) (A)

and MHCC-97H with ZNF143 knockdown (Mock/shNC/shZNF143#2/shZNF143#3)

(B) were collected at 0 and 24 h after releasing from synchronization with 2 mM

thymidine. The cell cycle distributions were examined by flow cytometry analysis.

DNA content was quantified using Modfit 3.2 software. Quantification of the cell

population in each phase is presented. C, The mRNA levels of S-phase genes in Li-7

and MHCC-LM3 cells with ZNF143 overexpression and in Hep 3B and MHCC-97H

cells with ZNF143 knockdown were analyzed by qPCR. For qPCR, values were

normalized to GAPDH values. Data are presented in fold change. D, The protein

expression of CDC6 was analyzed by Western blot after ZNF143 knockdown or

overexpression in HCC cells. E, Western blot assays were used to determine the

expression of ZNF143 and CDC6 after silencing CDC6 in HCC cells overexpressing

ZNF143. F, The protein levels of CDC6 and ZNF143 after overexpressing CDC6 in

ZNF143-knockdown HCC cells were measured by Western blot assays. G, MTT

assays were used to examine proliferation after silencing CDC6 in

ZNF143-overexpressing MHCC-LM3/Li-7 cells. H, Colony formation assays were

used to examine the colony formation capacity after decreasing CDC6 in

ZNF143-overexpressing MHCC-LM3/Li-7 cells. Bar graphs show the quantitative

analysis of colony numbers. I, MTT assays were used to examine proliferation after

overexpressing CDC6 in ZNF143-knockdown MHCC-97H/Hep 3B cells. J, Bar

graphs represent colony formation capacity after overexpressing CDC6 in

ZNF143-knockdown MHCC-97H/Hep 3B cells. The data are the mean of biological

triplicates and are shown as the mean ± SD. P values: *P < 0.05; **P < 0.01; ***P <

0.001 by two-tailed Student t test.

Figure 4. ZNF143 enhances CDC6 expression by activating MDIG. A and B,

ChIP-qPCR was performed to evaluate the enrichment of H3K9me3 in different

promoter regions of CDC6 in MHCC-LM3, Li-7 cells after overexpressing

ZNF143(A) and in MHCC-97H, Hep3B cells after decreasing ZNF143 (B). ChIP

enrichments were normalized to the input signal. C, qPCR was used to assess mRNA

alterations of histone lysine demethylases following ZNF143 overexpression. D,

Western blot analysis shows MDIG protein levels after ZNF143 overexpression and

knockdown in HCC cells. E, The cartoon indicates the sequence logo of ZNF143

potential binding site in JASPAR software (http://jaspar.genereg.net/). F, Wild-type

(wt) and mutated (mut) recognition sites of ZNF143 in the MDIG promoter region. G,

The relative activities of the MDIG promoter and the mutant promoter after

transfection of ZNF143 and Vector. (PWT: wild-type ZNF143 recognition site. PM1:

mutated ZNF143 recognition site1. PM2: mutated ZNF143 recognition site 2.). H,

ChIP assays with an anti-ZNF143 or negative control (anti-IgG) antibodies showed

ZNF143 binding to the recognition site 1 of MDIG promoter in MHCC-97H and Hep

3B cells. ChIP enrichments were normalized to the input signal. Error bars represent

the mean ± SD of at least three independent replicates. P values: *P < 0.05; **P <

0.01; ***P < 0.001 by two-tailed Student t test.

Figure 5. MDIG promotes HCC cell cycle progression by enhancing CDC6

expression. A, The left panel is a Venn diagram showing the overlap after silencing

MDIG in MHCC-97H and Huh7 cells. The numbers represent the number of altered

genes (fold change > 1.5, P < 0.05). The right panel shows KEGG analysis of the 66

overlapping downregulated genes in MHCC-97H and Huh7 cells with MDIG

knockdown in DAVID software. B, The protein level of CDC6 was analyzed by

Western blot assays after knockdown or overexpression of MDIG in HCC cells. C,

ChIP-qPCR was performed to evaluate the enrichment of H3K9me3 in different

promoter regions of CDC6 in MHCC-97H and Huh7 cells after knocking down

MDIG. D, ChIP-qPCR was performed to evaluate the enrichment of H3K9me3 after

MDIG knockdown in the promoter region of S phase genes. The PCR amplification

fragments for S phase gene promoter region near the transcription start site according

to UCSC (University of California, Santa Cruz) genome browser. The primers at +0.2

kb promoter regions of CDC6 were used to analysis. E, ChIP-qPCR analysis of

MDIG enrichment at the promoter of S phase genes in MHCC-97H and Huh7 cells. F,

Western blot showed the protein levels of CDC6 in MDIG-overexpressing HCC cells

after CDC6 knockdown. G, MTT assays were used to determine proliferation after

silencing CDC6 in MDIG-overexpressing HCC cells. H, Colony formation assays

were used to determine the colony formation ability after decreasing CDC6 in

MDIG-overexpressed HCC cells. Bar graphs show the quantitative analysis of colony

numbers. Error bars are the mean of biological triplicates and are shown as the mean

± SD. P values: *P < 0.05; **P < 0.01; ***P < 0.001 by two-tailed Student t test.

Figure 6. MDIG is required for ZNF143-mediated CDC6 activation. A and B,

Immunoblot analysis of ZNF143, MDIG, CDC6 and H3K9me3 in HCC cells after

ZNF143 overexpression (A) and quantified by densitometry (B). C and D, The

protein level of ZNF143, MDIG, CDC6 and H3K9me3 after knocking down ZNF143

were measured by Western blot (C) and quantified by densitometry (D). E and F,

Western blot analysis showed the protein levels of ZNF143, MDIG, CDC6, and

H3K9me3 in ZNF143-overexpressing HCC cells after knocking down MDIG (E).

The bands were quantified by densitometry (F). G, Li-7 cells stably overexpressing

ZNF143 with knockdown of MDIG were injected into one flank of nude mice.

Tumors were weighed. H, MTT assay was used to determine the proliferation after

silencing MDIG in ZNF143-overexpressing MHCC-LM3/Li-7 cells. I, Colony

formation assays were used to determine the colony formation ability after decreasing

MDIG in ZNF143-overexpressing MHCC-LM3/Li-7 cells. Bar graphs show the

quantitative analysis of colony numbers. Error bars are the mean of biological

triplicates and are shown as the mean ± SD. P values: *P < 0.05; **P < 0.01; ***P <

0.001 by two-tailed Student t test.

Figure 7. The clinical correlations of ZNF143, MDIG and CDC6 in HCC tissue.

A and B, ZNF143 mRNA level is positively correlated with the MDIG and CDC6

mRNA levels in TCGA dataset (A) and 30 HCC patients (B). For 30 HCC patients,

GAPDH was used as an internal control for qPCR analysis. C, Representative

immunohistochemical images of relative low (Case 1) versus high staining (Case 2) of

ZNF143, MDIG, and CDC6 expression in HCC tissues. Original magnification: upper

images, × 40; lower images, × 400. D, Correlations among ZNF143, MDIG, and

CDC6 protein levels in 211 human HCC tissues were examined by

immunohistochemistry. Number represents the number of tissue cases. h: higher

expression; l: lower expression. E, A schematic model of the ZNF143/MDIG/CDC6

axis showing it promotes HCC growth and cell cycle progression. By activating

MDIG, ZNF143 reduces the enrichment of H3K9mme3 on the CDC6 promoter region

and upregulates CDC6, which leads to enhanced proliferation of HCC. Data are

shown as the mean ± SD. P values: *P < 0.05; **P < 0.01; ***P < 0.001 by Pearson

correlation analysis.

ZNF143-mediated H3K9 trimethylation upregulates CDC6 by activating MDIG in hepatocellular carcinoma

Lili Zhang, Qi Huo, Chao Ge, et al.

Cancer Res Published OnlineFirst April 20, 2020.

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