Background
Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease with a 5-year survival rate of only 10% for all stages combined [
1]. According to GLOBOCAN 2020 estimates, PDAC is the seventh leading cause of cancer-related mortality worldwide and accounting for about 495,773 new cases and 466,003 deaths [
2]. Surgical resection remains the only treatment that offers a curative potential, whereas most cases lost the chance because of those presenting with advanced stages at the time of diagnosis [
3]. Currently, molecular targeted therapy showed great potential for improving the survival rates of PDAC patients [
4]. Then, it is significant for developing new therapeutic strategies and potential therapeutic targets for pancreatic cancer treatment.
WD repeat domain 3 (WDR3), also known as DIP2 or UTP12, belongs to the WD-repeat family and is a component of the 80 S complex of the small subunit processome, which is implicated in the 40 S ribosome synthesis pathway [
5]. It has been reported that WDR3 is involved in a variety of cellular processes including genome stability, cell proliferation, signal transduction, and apoptosis [
6‐
8]. McMahon et al proved that suppression of WDR3 reduced breast carcinoma cell proliferation and focus formation [
7]. Also, Akdi et al indicated WDR3 gene expression is associated with thyroid cancer risk in special populations [
9], and WDR3 can modulate genome stability in thyroid cancer patients [
10]. These studies revealed WDR3 confer growth and proliferative advantages of some malignant cancer, whereas the biological role of WDR3 in pancreatic cancer and the relevant mechanism remain unclear.
Studies proved the Hippo signaling pathway plays a critical role in modulating cell proliferation and has been demonstrated to contribute to the progression of malignant cancers, including pancreatic cancer [
11‐
14]. The core components of the Hippo signaling pathway, including yes association protein 1 (YAP1), promote the migration, invasion, and malignancy of cancer cells [
15], and inhibiting YAP1 expression suppresses pancreatic cancer progression by disrupting tumor-stroma interaction [
16,
17]. YAP1 usually enters the nucleus and interacts with other transcription factors, including TEA domain (TEAD) family members, to regulate downstream gene targets [
18‐
20]. Study of connective tissue growth factor (CTGF) and cysteine rich angiogenic inducer 61 (CYR61), the major downstream gene targets regulated by YAP1, has provided new insights into the physiological/pathological functions of Hippo pathway effectors [
12,
21]. CYR61 and CTGF had been reported to act as factors stimulating aggressiveness in a variety of cancers. CYR61 expression was exorbitantly higher in cancer cells and significantly triggered the aggressive phenotype in PADC [
22]. CTGF was a fibrosis-related gene related to pancreatic cancer progression by protecting pancreatic cancer cells from hypoxia-mediated apoptosis, and tumor cell-derived CTGF was vital for pancreatic cancer growth [
23]. Therefore, exploring methods to inhibit YAP1 expression is essential for improving pancreatic cancer therapy.
In our study, we proved that overexpressed WDR3 was correlated with poor survival in pancreatic cancer patients. Furthermore, WDR3 silencing could significantly decrease the proliferative and invasive abilities of pancreatic cancer cells by inducing YAP1 inhibition, which was found to rely on the interaction between WDR3 and GATA4. Taken together, our results emphasize the importance of WDR3 as a therapeutic target in pancreatic cancer.
Materials and methods
Cell culture
The PANC-1, MIA PaCa-2, and BxPC-3 cell lines were purchased from the Type Culture Collection Cell Bank of the Chinese Academy of Sciences (Shanghai, China). PANC-1 and MIA PaCa-2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (#30030, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (#10099141, Thermo Fisher Scientific) and 1% penicillin/streptomycin at 37 °C in a 5% CO2 incubator. BxPC-3 cells were cultured in RPMI-1640 medium (#88365, Thermo Fisher Scientific) supplemented with10% FBS and 1% penicillin/streptomycin at 37 °C in a 5% CO2 incubator.
Antibodies and chemicals
An anti-WDR3 antibody (#ab176817, working dilution 1:1000) was purchased from Abcam. An anti-GAPDH antibody (#10494–1-AP, working dilution: 1:3000), anti-GATA4 antibody (#19530–1-AP, working dilution: 1:1000), and anti-YAP1 antibody (#13584–1-AP, working dilution: 1:1000) were acquired from Proteintech. TED-347 (HY-125269, working concentration: 10 μM) was procured from MedChemExpress (USA).
Immunoprecipitation and western blot analysis
Whole cell lysates were obtained with RIPA lysis buffer (Cell Signaling Technology, Danvers, MA) containing 1% protease and phosphatase inhibitors (Sigma-Aldrich) on ice. The resulting cell lysates were centrifuged at 12,000 rpm for 15 min at 4 °C to remove undissolved impurities and collect the supernatants. The protein concentration was quantified using a BCA assay (#P0012S, Beyotime). Protein extracts (500 μg) were incubated with appropriate primary antibody beads overnight for an immunoprecipitation assay or directly evaluated for western blot analysis. The precipitated immune complexes were subjected to SDS-PAGE, transferred to 0.45-μm polyvinylidene difluoride (PVDF) membranes, and then immunoblotted with specific primary antibodies. The signal intensities of bands were quantified using ImageJ software.
Liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS) analysis
To identify potential WDR3-binding proteins, 293 T cells transduced with pcDNA3-WDR3 were collected for assays. WDR3 was pulled down by IP using an anti-WDR3 antibody and protein A + G agarose (#P2012, Beyotime) at 4 °C. LC-MS/MS analysis was performed using a Thermo Ultimate3000 liquid phase combined with Q Exactive Plus high-resolution mass spectrometry at Shanghai Applied Protein Technology. The data were retrieved with maxquant (v1.6.6) software, and the database retrieval algorithm was Andromeda. The database used in the search was the human proteome reference database of UniProt. The results were screened with a 1% FDR at the protein and peptide levels.
RNA-seq
A total amount of 1 μg of RNA per sample was used as the input material for RNA sample preparations. Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Clustering of the index-coded samples was performed on the cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform, and 150-bp paired-end reads were generated. FeatureCounts v1.5.0-p3 was used to count the read numbers mapped to each gene. Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1). We used the cluster Profiler R package to test the statistical enrichment of differentially expressed genes in KEGG pathways.
Quantitative RT-PCR assay
Total RNA was extracted with RNAiso Plus (#15596026, Invitrogen). The PrimeScript RT Reagent Kit (#RR047A, TAKARA, Japan) was used for reverse transcription. Real-time PCR (RT-PCR) was conducted with a TB Green™ Fast qPCR Mix kit (#RR430A, TAKARA, Japan). The 2-ΔCt method was used to quantify fold changes with normalization to GAPDH. Detailed information on the primer sequences is shown in Table S1.
RNA interference
Sh-Control and gene-specific shRNAs were procured from Sigma-Aldrich, and si-Control and gene-specific siRNAs were provided by RiboBio. Pancreatic cancer cells were transfected with siRNA using Lipofectamine 2000 (#11668019, Thermo Fisher Scientific) in accordance with the manufacturer’s instructions for 24 h, and then the Lipofectamine 2000-containing medium was replaced with fresh DMEM containing 10% FBS. 293 T cells were transfected with shRNA plasmids and packaging plasmids (pVSV-G and pEXQV) in Lipofectamine 2000 according to the manufacturer’s instructions for 24 h, and the Lipofectamine 2000-containing medium was replaced with fresh DMEM containing 10% FBS and 1 mM sodium pyruvate. At 48 h post transfection, the virus culture medium was collected and added to pancreatic cancer cells for 24 h of culture, after which the infected cells were selected with 1 μg/ml puromycin. The shRNA and siRNA sequences are shown in Table S2.
Chromatin immunoprecipitation (ChIP) and ChIP-qPCR
ChIP was performed with the Chromatin Extraction Kit (#ab117152, Abcam) and ChIP Kit Magnetic-One Step (#ab156907, Abcam) according to the manufacturer’s instructions. Purified DNA was analyzed using RT-PCR with a TB Green™ Fast qPCR Mix kit (#RR430A, TAKARA, Japan) following the manufacturer’s protocols. The ChIP-qPCR primers are shown in Table S3.
Nuclear and cytoplasmic extracts preparation
Cells were collected and the cell pellet was resuspended in 1 mL of Buffer A (10 mM HEPES-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40) to lyse the cells on ice for 10 min. Samples were spined down at 6500 rpm 4 °C for 3 min to pellet the nuclei. Nuclei pellet was washed with Buffer A and spined down at 3500 rpm for 5 min at 4 °C. The cell pellet was lysed by IP buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 1% protease inhibitor cocktails) on ice for more than 30 min. Protein concentration was determined by BCA protein quantification assay.
For colony formation assays, 500 pancreatic cancer cells transfected with sh-Control or sh-WDR3s were seeded in a six-well plate and cultured for approximately 10–12 days. Then, the colonies were fixed in methanol for 30 mins and stained with a 4 g/l crystal violet solution for 30 mins. The colonies were photographed, and the number of colonies was counted. All assays were performed in triplicate.
MTS assay
For MTS assays, transfected pancreatic cancer cells were seeded in 96-well plates with 2500 cells per well. After 72 h of culture, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS reagent) (Abcam, #ab197010, USA) was added to each well for three hours of culture according to the manufacturer’s instructions. The absorbance in each well was measured with a microplate reader at 490 nm. Each experiment included five replicates and was performed in triplicate.
Cell invasion assay
Cell invasion assays were performed using transwell chambers (8-μm pore size; Millipore) with a Matrigel (BD Biosciences, CA, USA) matrix. In brief, 600 μl of complete medium supplemented with 30% FBS was added to the bottom chamber, and 105 transfected pancreatic cancer cells were suspended in 200 μL of serum-free medium and added to the upper chamber. After culturing for 12–24 h, the cells on the top surface of the membrane were mechanically removed using a cotton swab. The cells on the bottom surface of the membrane were fixed in methanol for 30 mins and stained with a 4 g/l crystal violet solution for 30 mins. The invaded cells were counted under a microscope, with five fields per well evaluated. Each experiment was performed in triplicate.
Gene correlation analyses between the mRNA expression levels of WDR3 and YAP1 were carried out with the GEPIA database (
http://gepia.cancer-pku.cn/) for all given sets of GTEx and TCGA expression data. The Eukaryotic Promoter Database (
https://epd.epfl.ch//index.php) was used to determine the potential binding sites of GATA4 in the promoter of the YAP1 gene.
PDAC xenografts in nude mice
Animal experiments were approved by the Ethical Committee on Animal Experiments of the Sichuan Provincial People’s Hospital in Chengdu, China. PANC-1 cells (3 × 106) infected with sh-Control or sh-WDR3 #1 were subcutaneously injected into the left flank of BALB/c-nu mice (4–5 weeks old, male) purchased from Vital River. Tumor sizes were assessed with a digital Vernier caliper every three days. Tumors were harvested 3 weeks after injection, and tumor weights were measured.
Orthotopic syngeneic model of pancreatic cancer to C57BL/6 mice
We used 8-week-old wild-type C57BL/6 mice in the experiments. For orthotopic implantation, mice were anesthetized with pentobarbital sodium, and hair was removed from their abdomens. We incised each mouse longitudinally along the abdomen to expose the pancreas, injected 20 μL of the cell suspension into the pancreas, and closed the incision with sutures. Each experimental group consisted of five mice. All mice were weighed and checked for signs of distress regularly. Abdominal palpation was used to monitor tumor size. Tumors were harvested 3 weeks after injection, and tumor weights were measured.
Statistical analysis
All data are expressed as the mean ± standard deviation (SD) of three independent experiments. Comparisons between two groups were performed using Student’s t-test, and two-way ANOVA or one-way ANOVA together with the Bonferroni post hoc test was used for multigroup analysis. A P value less than 0.05 was considered significant. GraphPad Prism 6 software (GraphPad Software, Inc.) was used for statistical analysis.
Discussion
In our study, we first proved that WDR3 was overexpressed and positively correlated with poor survival in pancreatic cancer (Fig.
1). Akdi et al reported that WDR3 is overexpressed and a risk factor in thyroid cancer [
9]. Additionally, several groups have also reported the biological role of WDR3 in modulating genome stability [
10], increasing cancer predisposition [
26], promoting proliferation and arresting cancer cells in the G1 phase of the cell cycle [
7]. Consistently, our results also indicated that overexpressed WDR3 increased the proliferation and invasion abilities of pancreatic cancer cells (Fig.
2). However, the biological mechanism of WDR3 overexpression in pancreatic cancer still needs further study.
GATA binding protein 4 (GATA4), a protein in the GATA family of zinc-finger transcription factors, can recognize the GATA motif, which is present in the promoter of many tumor-related genes. GATA4 regulates the expression of genes involved in multiple pathological/physiological processes, including embryogenesis, myocardial differentiation and function, and normal testicular development. It has been reported GATA4 expression is associated with increased tumor size, metastasis, and a poor prognosis [
27]. GATA4 mRNA expression is upregulated in pancreatic cancer cell lines and tissues [
25], and downregulation of GATA4 expression increases drug sensitivity in cancer cells [
28]. GATA4 can decrease P53 protein expression by transcriptionally activating the expression of MDM2 [
29], the primary negative regulatory factor of the P53 protein that induces p53 ubiquitination and degradation [
30]. GATA4 is also highly expressed in most hepatoblastomas and correlates with a mesenchymal, migratory phenotype in hepatoblastoma cells by regulating the expression of ADD3, AHNAK, and IGFBP1 [
31]. Similarly, our results indicated that GATA4 could function as a transcription factor to induce YAP1 expression and activate the Hippo signaling pathway, which resulted in pancreatic cancer progression. GATA4 knockdown reversed the inhibition of YAP1 and proliferation and invasion of abilities of pancreatic cancer cells induced by WDR3 silencing and reversed the upregulation of YAP1 expression induced by WDR3 overexpression (Fig.
6). Taken together, our results provide new insights into the specific mechanism by which GATA4 regulates the progression of pancreatic cancer.
The Hippo signaling pathway was first discovered in studies of
Drosophila melanogaster [
32]. Hippo signaling governs normal organ development and tissue regeneration under physiological conditions [
33]. Hippo signaling is an evolutionarily conserved network that plays a key role in regulating cell proliferation, organ growth, and regeneration [
34]. YAP1 is the key downstream regulator in the Hippo pathway that exhibits upregulated expression in pancreatic cancer [
35‐
37]. Aberrant transcriptional activity of YAP1 has crucial roles in pancreatic tumor cell biology, including roles in growth, epithelial-mesenchymal transition (EMT), microenvironmental signaling transduction, and drug resistance [
34]. Then, inhibition of YAP1 expression is essential for pancreatic cancer targeted therapy. Studies have shown that YAP1 expression is induced by KRAS activation [
35], aerobic glycolysis [
38], GNAS [
39], and the cancer upregulated gene (CUG) 2 exhibiting upregulated expression in lung cancer which could increase the expression of YAP1 [
40]. Interestingly, our results identified a protein interaction between WDR3 and GATA4 that led to the regulation of GATA4 nuclear translocation and YAP1 expression in pancreatic cancer. Silencing WDR3 significantly decreased the expression levels of YAP1 and the downstream target genes CTGF and CYR61 in pancreatic cancer. All these results emphasized the clinical significance of WDR3-targeted therapy.
TED-347 is a potent, irreversible, covalent, allosteric inhibitor of the YAP-TEAD protein-protein interaction [
24]. TED-347 forms a covalent complex with TEAD4 that inhibits TEAD4 binding to YAP1, blocks YAP1 transcriptional activity, and suppresses the expression of downstream target genes, including CTGF and CYR61. Combined with the inhibition of YAP1 transcription induced by WDR3 knockdown, TED-347 treatment further enhanced the ability of WDR3 silencing to inhibit pancreatic cancer progression.
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