Background
DNA methylation is an important mechanism of epigenetics, which alters gene expression without changing the sequence of DNA. The members of the ten-eleven translocation family (TET1, TET2, and TET3) of dioxygenases play a key role in DNA demethylation by catalyzing the hydroxylation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) [
1‐
3] and regulating the methylation levels of 5-cytosine (5-C) in coordination with DNA methyltransferases (DNMTs) [
4]. Enrichment of 5-hmC and decrease of 5-mC in promotor CpG islands are considered to be indicators of gene activation [
5]. The 5-hmC content in CpG islands is mainly regulated by the TET family [
1,
3]. Abnormal expression or gene mutations of members of the TET family have been reported in various kinds of cancers in humans. It was reported that aberrant expression of TET1 was more frequent in solid tumors [
6‐
8],
TET2 was frequently mutated in hematopoietic tumors [
9], while TET3 remains less discussed [
10].
TET1 was first found in a patient with a rare variation of t (10, 11)(q22;q23) acute myeloid leukemia (AML), and the gene is located at chromosome 10q21.3 [
11]. With the exception of aberrant expression in cancer progression, TET1 is involved in cell differentiation [
12,
13]. The role of TET1 in gastric [
6], breast [
14], hepatic [
7], prostate [
8], and colon [
15] cancer is well-studied. In these tumors, TET1 is mostly considered to be a tumor suppressor, while it is also reported as an oncogene [
16]; it binds directly to the promoters of anti-oncogenes, catalyzes 5-mC hydroxylation to initiate demethylation, and induces target genes involved in transcriptional activation, such as phosphatase and tensin homolog (PTEN) in gastric cancer [
6].
Pancreatic tumors are among the most lethal malignant tumors. Unlike the steady increase in survival rates for most cancers, the 5-year survival rate of patients with pancreatic tumors is 8%; furthermore, the 5-year survival rate for patients diagnosed with advanced stages of pancreatic cancer is only 2% [
17]. Aberrant DNA methylation in pancreatic tumors has been widely studied in recent years [
18,
19], while the relationship between TET1 and pancreatic cancer is still unclear, with only a decrease in 5-hmC levels reported by Yang [
20]. In this study, we determined the potential involvement of TET1 in pancreatic cancer progression, focusing on tumor proliferation and metastasis.
We found TET1 was downregulated in pancreatic tumor tissue compared to adjacent non-tumor tissue in > 3/5 pancreatic cancer patients, and low expression of TET1 was associated with shorter overall survival. We utilized RNA arrays to demonstrate that overexpression of TET1 inhibited pancreatic tumor epithelial-mesenchymal transition (EMT) by suppressing the Wnt/β-catenin signaling pathway, but not the Transforming growth factor (TGFβ) or NOTCH signaling pathways. Secreted frizzled-related protein 2 (SFRP2), the upstream inhibitor of the Wnt/β-catenin pathway, was transcriptionally activated by overexpression of TET1, which increased 5-hmC content in the SFRP2 promoter. We also revealed that TET1 inhibited proliferation of pancreatic tumors by inducing GO/G1 arrest in the cell cycle, although the underlying mechanism requires further study. This is the first report of TET1 as a suppressor of pancreatic tumors, further elucidating the role of TET1 in pancreatic cancer.
Materials and methods
Pancreatic tumor cell lines and tissue samples
Cell lines were obtained from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Sw1990 and BXPC3 cells were cultured in Dulbecco’s Modified Eagle’s Medium and RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), respectively, supplemented with 10% fetal bovine serum (FBS, Biological Industries, Beit-Haemek, Israel), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Gibco) at 37 °C in humidified air with 5% CO2. A total of 15 paired pancreatic tumor and adjacent non-tumor tissue samples were collected from Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. The pancreatic tissue microarray was purchased from Shanghai Outdo Biotech, Shanghai, China. All patients included in the analysis were treated by R0 resection, and didn’t received radiotherapy, chemotherapy and neoadjuvant therapy.
Plasmids and stable cell lines
The full-length human TET1 overexpression plasmid (TET1-OE) was obtained from Addgene (Cambridge, MA, USA). We also constructed a TET1-CD-mutated plasmid (TET-MUT) by modifying amino acids 1672 H to Y and 1674 D to A. The vector backbone pEF1a (Invitrogen, Carlsbad, CA, USA) was set as the empty vector (EV). The plasmids were transiently transfected into the SW1990 and BXPC-3 cell lines with Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol.
Stable TET1 knockout cell line SW1990-KO was constructed by CRISPR/CAS9. The lentivirus packed with pGMLV-CA1 expressing Caspase-9 and lentivirus packed with pGMLV-GM2 expressing gRNA targeting the sequence 5′-CAGCACGCATGAATTTGGAT-3′ in the TET1 sequence were constructed by Genomeditech, Shanghai, China. We conducted selection of the monoclonal TET1 knockout cell line in our laboratory. We also built a SW1990 control cell line SW1990-NC cell line by transfected SW1990 only with the lentivirus packed with pGMLV-CA1 expressing Caspase-9.
The stable TET1 overexpressing cell line BX-TET1-OE was generated using the dCAS9-SAM system. Lentiviruses expressing dCAS-VP64, MS2-P65-HSF1, and gRNA, respectively were constructed by Genomeditech. The TET1 overexpressing gRNA was designed to target the sequence 5′-AGGGGGTCGAGAGGGAGTCG-3′. We also built a BXPC-3 control cell line BX-TET1-NC cell line by transfected BXPC-3 with the dCAS9-SAM system, which gRNA was designed to target the sequence 5′- GTTCTCCGAACGTGTCACGT-3′.
SFRP2 siRNA was designed as follows: Si-SFRP2–1 5′-GTGAGGAGATGAACGACAT-3′, Si-SFRP2–2 5′- GCAAGACCATTTACAAGCT-3′, Si-SFRP2–3 5′- GCATCGAATACCAGAACAT-3′.
RNA extraction and RT-PCR analysis
Total RNA was extracted with TRIzol® (Invitrogen) and reverse-transcribed using a cDNA reverse transcription kit (Toyobo Life Science, Shanghai, China). The cDNA was diluted 1:10 in DNase and RNase-free dH
2O. Real-time PCR was performed with an ABI 7900 instrument using SYBR Green PCR Master Mix (Toyobo Life Science). Quantification was determined by the delta-delta Ct method, expressed in arbitrary units, and normalized to GAPDH. The primers used are listed in Additional file
1: Table S1.
DNA extraction and dot blot
Genomic DNA of tissues and cell lines was extracted using a DNA isolation kit (Tiangen Biotech, Beijing, China). The DNA was denatured at 99 °C for 5 min, then cooled on ice. Subsequently, DNA was spotted onto a nitrocellulose membrane and air-dried. The DNA was UV-cross-linked to the membrane, which was then blocked in 5% non-fat milk in TBST, incubated with anti-5-hmC or anti-5-mC antibody (GeneTex, Irvine, CA, USA) at 4 °C overnight. The membrane was then incubated with anti-mouse IgG-HRP secondary antibody. Finally, the membrane was visualized by ECL (Beyotime Biotechnology, Shanghai, China).
Protein extraction and western blotting
Total protein was extracted with RIPA buffer containing PMSF and sonicated. Nuclear protein and cytoplasmic protein were extracted with the Nuclear-Cytosol Extraction Kit (Beyotime Biotechnology). Protein samples were separated by SDS-PAGE and transferred onto PVDF membrane and blocked with 5% BSA in TBST, washed with TBST, incubated with the primary antibody at 4 °C overnight. The membrane was washed and incubated with HRP-conjugated secondary antibody at room temperature for 2 h. The membrane was washed again and visualized by ECL. TET1 antibodies were purchased from GeneTex. GAPDH, GSK-3β, phospho-GSK-3β (Ser9), p16, and EMT antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
BXPC-3 cells were cultured in 6-well plates and transfected with 5 μg of TET1-OE, TET1-Mut, or EV controls using Lipofectamine 2000 according to the manufacturer’s protocol. After 48 h, cells were harvested and seeded at 2000 cells/well in 6-well plates for 14 days. Colonies were then stained with gentian violet (ICM Pharma, Singapore) and counted.
Cell proliferation assay
The transiently transfected BXPC-3, SW1990-KO, and SW1990-NC cells were seeded at 2000 cells/well in 96-well plates. Proliferation was detected with a Cell Counting Kit-8 (Dojindo, Shanghai, China) after 24, 48, and 72 h, measured by OD450.
Wound healing and transwell migration assays
The 3 transiently transfected BXPC-3 cell lines, SW1990-KO, and SW1990-NC were cultured in 6-well plates until confluent. The monolayers were scratched, washed with PBS, cultured in serum-free RPMI 1640, and viewed after 0, 24, and 48 h.
For the transwell migration assay, we used Corning Transwell chambers (Corning Life Sciences, Corning, NY, USA). For migration, 2 × 105 cells suspended in 200 μL serum-free medium were added to the upper chamber with 400 μL medium containing 10% FBS added to lower chamber, and the chambers were cultured for 24 h. For invasion, the upper chamber was coated with 20 μg Matrigel (BD Biosciences, San Jose, CA, USA) and cultured for 48 h using the protocol described above. Subsequently, the cells that migrated through the membrane were fixed and stained with crystal violet, and counted in 3 random fields.
Flow cytometry analysis
Stable TET1-overexpressing BX-TET1-OE, knockout SW1990-KO, and their negative control cell lines were cultured in 6-well plates until 70% confluent and starved overnight. RPMI 1640 containing 10% FBS was added to the wells. After 6 h, the cells were harvested and fixed in ice-cold 70% ethanol overnight at 4 °C, and mixed with propidium iodide (BD Pharmingen, BD Biosciences) for 30 min at 37 °C in the dark. Data were analyzed with CellQuest software (BD Biosciences).
Immunohistochemical staining (IHC)
The tissue samples and a tissue microarray including 63 pancreatic tumor tissues (57 of 63 had their adjacent non-tumor tissues) purchased from Shanghai Outdo Biotech were collected for IHC staining. The tissue samples of tumor xenograft mice were collected three regions per tumor. The tissue sections were de-waxed, hydrated, incubated in citrate buffer for antigen retrieval and fixed, then blocked in TBST containing 5% BSA. Tissue sections were incubated with anti-TET1 antibody at 4 °C overnight, washed in TBST 3 times for 10 min and incubated with anti-mouse IgG-HRP secondary antibody for 2 h at room temperature, then washed again in TBST.
Immunofluorescence staining
BX-TET1-OE and BX-TET1-NC cells were incubated on coverslips in 6-well plates, and some were transfected with siRNA siSFRP2 for 48 h. Cells were then fixed in 4% paraformaldehyde and incubated with β-catenin primary antibody at 4 °C overnight and fluorochrome-labeled anti-rabbit IgG secondary antibodies. Cells were then stained with DAPI and subsequently viewed by confocal microscopy.
RNA array analysis
EMT RT
2 Profiler PCR Arrays were purchased from Qiagen (Hilden, Germany). RNA extracted from BX-TET1-OE, BX-TET1-NC, SW1990-KO and SW1990-NC cell lines were reverse-transcribed into cDNA and added to the RT
2 Profiler PCR Array plates following the manufacturer’s protocol. The results were analyzed by Qiagen’s online tool (
https://www.qiagen.com/).
GlucMS-qPCR to determine 5-hmC and 5-mC content
We performed GlucMS-qPCR to detect 5-hmC and 5-mC content in promoter regions using the EpiMark® 5-hmC and 5-mC Analysis Kit (New England Biolabs, Hitchen, UK). Genomic DNA was treated with T4 Phage β-glucosyltransferase and UDP-Glucose to modified 5-hmC sites to 5-ghmC, and then digested by MspI and HapII following the manufacturer’s protocol. MspI cleaves 5-hmC and 5-mC, but not 5-ghmC, while HapII cleaves 5-hmC, 5-mC, and 5-ghmc. Then, we analyzed the enzyme-digested product by semi-quantitative PCR. The primers used to determine 5-mC and 5-hmC content in the promoter of SFRP2 were: forward 5′-TAGGATTTCTTTAAACAACAAACAGAGAA-3′ and reverse 5′-ATGCCTGGCAACCCAGCAGAAACT-3′.
Bisulfate sequencing
Genomic DNA was bisulfate-treated using the EpiTect Bisulfite Kit Qiagen following the manufacturer’s protocol. Bisulfate-treated DNA was amplified by PCR with the following primers: SFRP2 Forward: 5′- TTTTTTACGGTATTGGGGAGTATAT-3′ and Reverse: 5′-CCGAAATTTCTACTAAATTACCAAAC-3′. PCR products were then cloned into the T-Easy Vector (Quanshijin Biotechnology, Beijing, China). For each treated group, 10 clones were randomly selected for sequencing, and the results were analyzed by QUMA, an online CpG methylation analysis tool (
http://quma.cdb.riken.jp/).
Chromatin immunoprecipitation (ChIP)
ChIP was performed in BXPC-3 cells. Cells cultured in 10 cm plates were cross-linked with formaldehyde and processed following the ChIP kit protocol (EMD Millipore, Burlington, MA, USA). The target DNA fragments were detected by PCR. The primers used to detect target DNA fragments are as follows: SFRP2-Forward 5′-AAACAGAGAAGCCTGGCCG-3′, SFRP2-Reverse 5′-TTCGGACTGGGGCAAAACAA-3′.
Animal model of tumor xenograft
BALB/c nude mice (6-weeks old, male) were purchased from the Chinese Academy of Sciences.
(Shanghai, China) and kept in a specific pathogen-free environment.
For subcutaneous tumorigenesis, BX-TET1-OE and negative control cell line BX-TET1-NC were harvested and suspended in 1 × PBS. A total of 3 × 106 cells were injected into mice subcutaneously.
For in vivo orthotopic implantation of the pancreatic cancer model, BX-TET1-OE and BX-TET1-NC cells were labeled with firefly luciferase, with 1 group of BX-TET1-OE being transfected with siSFRP2 for 48 h before injection. Then 3 × 10
6 cells suspended in 100 μL medium (containing 1/3 Matrigel) were injected into the pancreata of mice. An additional abdominal injection of 200 nmol/kg si-SFRP2 was performed in si-SFRP2 group after 1 week, while 200 nmol/kg si-NC were injected into other two groups. Metastasis was photographed using a Xenogen noninvasive bioluminescence In Vivo Imaging System (IVIS, PerkinElmer, Waltham, MA, USA) as described previously [
21].
Mice were euthanized after 3 weeks. The implanted tumors and metastatic foci were observed by general observation, H&E staining, and IHC staining. The pancreatic tumor and liver metastasis samples of tumor xenograft mice were collected three regions per tumor and liver.
Statistics
Data analysis was performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA), and data are presented as the mean ± SD from at least 3 separate experiments. Kaplan–Meier method was used for overall survival. Student’s t-test, Kendall’s tau-b, and Spearman correlations were also used. In Kendall’s tau-b correlation analysis, the dichotomous variables: 1, − 1 were used instead of levels of 5-hmC, TET1, TET2, TET3 in tumor tissues higher in non-tumor tissues or the opposite, respectively. Statistical significance was set as P < 0.05.
Discussion
As the major form of DNA epigenetic modification, 5-mC is modified in > 60% CpG sites in the mammalian genome, and plays an important role in differentiation and disease [
26]. Members of the TET family convert 5-mC to 5-hmC, initiating DNA demethylation, and ultimately generating 5-C with the help of thymine DNA glycosylase (TDG) and base excision repair (BER) [
3]. Recently, TET-mediated DNA demethylation was widely studied in pluripotency, differentiation, and cancers [
6‐
8,
13,
14]. In this study, we first demonstrated the involvement of TET1 in pancreatic tumors. We found TET1 was downregulated in pancreatic tumor tissues and cell lines, and that pancreatic tumor patients with low TET1 levels have shorter overall survival than patients with high levels of TET1. TET1 suppressed pancreatic tumor proliferation, migration, and invasion in vivo and in vitro via inhibiting the Wnt/β-catenin signaling pathway, which is consistent with results found for colon and ovarian cancer [
27,
28].
EMT can be induced by various signaling pathways (TGF-β, Wnt/β-catenin, Notch, FGF, EGF, and HGF) and hypoxia in tumor progression [
29]. Here, we performed RNA array analysis to determine the relevant EMT signaling pathway, specifically identifying the Wnt/β-catenin pathway. Although mRNA expression of JAG1 and NOTCH1 in the NOTCH pathway were upregulated in the TET1 overexpressing group, the NOTCH pathway is considered to be an activator of EMT [
29], and cross-talk between the NOTCH and Wnt/β-catenin pathways did not support the phenotype in our study; thus, we focused on the Wnt/β-catenin pathway. In pancreatic tumors, we revealed that TET1 diminished β-catenin in both the nucleus and cytoplasm and disturbed the intracellular migration of active β-catenin. Levels of cyclin D1, a downstream target of β-catenin, were also decreased. These results provide strong evidence that TET1 suppresses EMT in pancreatic tumors by inhibiting Wnt/β-catenin.
Wnts bind to FZD receptors and lipoprotein receptor-related protein 5/6 (LRP5/6) co-receptors to form a complex that initiates canonical Wnt signaling (β-catenin-dependent signaling). The mRNA levels of Wnt pathway-associated genes, including
CTNNB1,
FZD7,
WNT11,
WNT5A, and
WNT5B, were found to be downregulated, and
GSK-3B was upregulated.
CTNNB1 encodes β-catenin,
GSK-3B encodes GSK-3β, and both are key in the Wnt/β-catenin pathway. The
FZD7 gene encodes Frizzled-7, a member of the Frizzled family, is a receptor of Wnt signaling [
30], and has been reported to be an oncogene in various cancers [
22,
23].
WNT11,
WNT5A, and
WNT5B belong to the non-canonical Wnt signaling pathway (β-catenin-independent signaling), and are involved in oncogenesis and in several developmental processes [
31‐
33]. Expression of these oncogenes were consistently suppressed in our study, verifying the inhibition of both canonical and non-canonical Wnt signaling. As antagonists of the Wnt pathway, DKKs and SFRPs competitively bind the Wnt-binding site of Frizzled proteins to modulate Wnt signaling [
34]. In our study, DKKs and SFRPs were all upregulated in response to TET1 overexpression except DKK1, which is a similar result obtain by two other studies in colon and ovarian cancer [
27,
28]. The level of SFRP2 mRNA was elevated by ~ 70-fold. Following the classical molecular mechanism of TET1-mediated transcriptional activation, we demonstrated that TET1 binds directly to the SFRP2 promoter, catalyzes 5-mC hydroxylation to 5-hmC in the promoter CpG islands, induces demethylation, initiates SFRP2 transcriptional activation, and ultimately inhibits EMT in pancreatic tumors. Except TET1, other molecular mechanisms inhibiting Wnt signaling in pancreatic tumor like FOXO1-related LINC01197 has been reported [
35], these mechanisms may contributes the final activity of Wnt signaling pathway in pancreatic tumor.
In our study, the inhibitory function of TET1 in pancreatic tumor proliferation was detected in vivo and in vitro. Arrest of the cell cycle in G0/G1 phase indicates the cell cycle-associated proteins cyclin D1 and p16 may be involved in this mechanism; however, this this needs to be further investigated. As anti-oncogenes, p16, RASSF1A, and ppENK have been associated with aberrant hypermethylation in pancreatic tumors [
24,
25]. Previous studies have shown that p16 methylation is correlated with an increased risk of pancreatic cancer [
36], and DNA hypermethylation of the promoter was observed in 60% of p16 genes, and was markedly correlated with decreased mRNA expression [
37]. The ppENK gene methylation level was found to be increased by < 90% in pancreatic cancer [
24]; methylated ppENK was detected in all pancreatic cancer cell lines tested, and was associated with loss of mRNA expression in pancreatic carcinoma cell lines and normal pancreatic tissues [
38]. RASSF1A hypermethylation was detected in 29 out of 45 (64%) primary adenocarcinomas, 10 out of 12 (83%) endocrine tumors, and 8 out of 18 (44%) pancreatitis samples [
25]. Therefore, we investigated expression of these genes, and found the expression of ppENK and RASSF1A were not influenced by TET1, only p16 was upregulated at the mRNA and protein levels. As an inhibitor of cyclin-dependent kinases (CDK), p16 slows down the cell cycle by prohibiting progression from G1 phase to S phase [
39]. The upregulation of p16 may have contributed to the G0/G1 arrest observed in our study; however, the underling mechanism remains to be elucidated.
Targeted therapy is a therapeutic strategy with more effective treatments and reduced toxicity, but the limited knowledge of potential targets suppresses its application scope in tumor patients [
40]. For therapy of pancreatic tumor patients with TET1 low expressing, our finds provide potential targets including TET1 and its downstream gene SFRP2 for therapy. CRISPR–Cas9-based genome editing provides a new therapeutic strategy with high specificity [
41], such as the dCas9-multiGCN4/scFv-TET1CD-sgRNA-based SFRP2-targeted demethylation system provides a SFRP2-targeting therapeutic strategy to inhibit pancreatic tumor metastasis [
42]. With the development of technology, more novel targeted therapeutic strategy will be available.
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