#Authors contribute equally to this work
Endometrial cancer remains to be a major type of malignancy in threatening female life. Molecular insights in advancing our understanding of endometrial tumorigenesis are much needed. We here report that a less-studied protein Dihydropyrimidinase like 3 (DPYSL3) is a potent tumor suppressor. DPYSL3 is uniquely regulated by wild type p53 (wtp53), and its expression is at the highest level when cells carry wtp53 and are exposed to hypoxia. We reveal that wtp53 can bind DPYSL3 promoter to enhance DPYSL3 expression and in turn, the elevated DPYSL3 can restrain cancer cell proliferation and invasion
Endometrial cancer (EC) as one of the most common type of malignancy in female exhibits a rapid growth of morbidity around the world in recent years (
Human solid tumors are invariably less well-oxygenated than the normal counterpart tissues from which they arose, albeit with variable incidence and severity within a given patient population. This so-called tumor hypoxia is a negative prognostic and predictive factor due to its resistance to radiotherapy and anticancer chemotherapy as well as predisposing for increased tumor metastases. Evidence from clinical and experimental studies increasingly point out that tumor hypoxia exert a variety of influences on activation of certain signal transduction pathways, gene regulatory mechanisms and induction of processes for DNA damage, tumor apoptosis and angiogenesis (
Under conditions of hypoxia-induced stress, p53 is stabilized by the ATR and ATM kinases and facilitated by MDM2 reduction (
Our previous RNA sequencing study revealed a novel gene, Dihydropyrimidinase like 3 (DPYSL3), highly expressed in tetracycline-induced p53 TET-on system in COLO-684 endometrial cancer cell line compared to un-induced control (data not shown). DPYSLs family (DPYSL1-5) is majorly highly expressed in nervous system and plays an essential role in neurite outgrowth, guidance and axonogenesis during neural development but generally decrease in adult brain (
All cell lines used in this study were obtained from Type Culture Collection of the Chinese Academy of Sciences (Beijing, China). Flag, DPYSL3, MEK, ERK, pERK and GAPDH primary antibodies were purchased from SAB Biotech (College Park, MD, USA). Deferoxamin and 5-FU were from Sigma-Aldrich (Billerica, MA, USA). DPYSL3 siRNA (5’-GGAUAAUACUCUACCACCAT-3’) was synthesized from Genepharma, Shanghai, China. DPYSL3 cDNA was cloned into pIRES2-EGFP-SF plasmid (Vigene Biosciences, Jinan, China). The vectors of truncating DPYSL3 were prepared based on wild type DPYSL3 vector using mutagenesis PCR. The following PCR primer sequences were listed in
The ChIP assay was performed as previously described (
In brief, six genomic DNA fragments containing p53 binding sites of DPYSL3 (CBS1-4) were cloned into pGL3-promoter-reporter (Promega), respectively, and mixed with pcDNA3-wtp53 or mtp53 (R248G) as well as pRL-TK (Renilla luciferase-expressing construct; Promega), then co-transfected into Calu-6 cells. 48 h later, the Renilla-luciferase substrate coelenterazine (2.5 μg/mL) was added into the plates to detect the luciferase activity as a measure of transfection efficiency. The firefly luciferase substrate D-luciferin (100 μg/mL) was added for detection of p53-driven reporter activation.
TRIzol (Thermo Fisher Scientific) was used to extract total RNA according to the manufacturer’s instructions. cDNA synthesis was performed using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). All qPCR was performed using SYBR Premix ExTaq Kit (Roche, Basel, Switzerland) to analysis mRNA. The primers were listed in
1 × 105 cells after 48-h transfection were plated in 96 well plate, followed by adding 10 μL CCK-8 solution (Solarbio, Beijing, China) to incubate additional 1 h. Absorption values of 450 nm were examined using Multiskan FC microplate reader (Thermo Fisher Scientific) to construct the regression equation and calculate IC50. The IC50 values were evaluated the cell proliferation.
1 × 105 cells were cultured within 200 μL suspension in upper and 800 μL fresh medium in lower transwell chamber (Corning, New York, NY, USA) on 24-well plates for 24 h. The cells at the lower chamber were cross-linked by 1% paraformaldehyde for 10 min and stained by 0.5% crystal violet (Sangon Biotech, Shanghai, China) for 5 min. The stained cells were counted under Olympus BX-51 microscope to evaluate the cell invasion.
1 × 105 cells were sub-cultured in 6 well plate with 80% density, and a straight line was gently created using a p200 pipette tip. Cells were then washed with PBS and cultured in medium for the period of the assay. After 8-h and 24-h cell culture, the distance between the two sides of the scratch was captured, measured the dynamic change of width and analyzed the cell migration by Image J. Amount of scratch closure was calculated as previously described (
Transfected with appropriate plasmids and empty vectors used as the experimental controls accordingly, the whole cell lysates or nuclear extracts were mixed 1 μg Flag, MEK or Rabbit IgG antibody, and 40 μL flurry IgA beads (Invitrogen) for rotating overnight at 4°C. Immunoprecipitates were washed by IP buffer (20 mM HEPES [pH 7.9], 350 mM NaCl, 0.1% NP-40, 1 mM DTT, 0.2 mM PMSF, 2 mg/mL leupeptin and 2 mg/mL aprotinin) and western blotted for appropriate antibodies. For immunoblotting, the transfer-ready membranes were blocked overnight in TBS (10 mM Tris-HCl [pH 7.5], 150 mM NaCl) containing 5% nonfat milk and 0.1% Tween-20 at 4°C, followed by incubation with appropriate primary antibodies. The secondary antibodies of horseradish peroxidase-conjugated anti-mouse, -rabbit, and -goat antibodies were used at a 1:5000 dilution.
Animal experiments in the current study were approved by our Institutional Laboratory Animals Committee and conducted following The Guidelines for Laboratory Animal Care and Use by the Affiliated Yantai Yuhuangding Hospital of Qingdao University. A total 16 BALB/c nude mice (male, weight 20–25 g) were supplied by Shanghai SLAC Laboratory Animal (Shanghai, China). Xenograft experiments were performed as described previously (
The full length of amino acid of DPYSL3 and MEK were achieved and input into I-TASSER server (
Data are presented as means ± standard deviations for three independent experiments. The difference of values was analyzed using Student’s
Commercialized kits, reagents, instruments, software, antibodies, etc., used in the research, shall be provided with their full name, along with the information of the Manufacturers/suppliers/software details (Name, City, Province/State, Country).
Accession numbers of RNA, DNA and protein sequences used in the manuscript should be provided.
To validate the presence of DPYSL3 up-regulation by p53 over-expression in our previous RNA sequencing data, DPYSL3 mRNA and protein level were examined in EC cells of COLO-684 and HEC-108 (wild type p53, wtp53) as well as Ishikawa and HEC-59 (mutated p53, mtp53) treated with a hypoxia-mimic drug deferoxamine (DFO) or 5-fluorouracil (5-FU) known to damage DNA. The higher expression of DPYSL3 was observed after DFO or 5-FU exposure compared to non-treatment both in COLO-684 but not in Ishikawa cells (
The mRNA and protein levels of DPYSL3 normalized by GAPDH in COLO-684 and HEC-108 (wild type p53) (A,B) as well as Ishikawa and HEC-59 (mutant p53) (C,D) before and after DFO or 5-FU treatment. Data are presented as the mean ± SEM of three individual experiments. * and # represent the comparison between DFO or 5-FU and control group with
As a transcription factor, p53 has been well revealed its DNA binding domain sequence (
The luciferase activity derived from the promoter of DPYSL3 with different truncating p53 consensus binding sequences in EC cells (A). The enrichment of p53 on the promoter of DPYSL3 in EC cells (B). Data are presented as the mean ± SEM of three individual experiments. In panel A, * represents the comparison between CBS and blank groups, and # represents the comparison between Ishikawa/HEC-59 and COLO-684/HEC-108 groups with
Now that DPYSL3 has been demonstrated to be promoted by wtp53 under hypoxia condition in EC cells, therefore, the potential effect of DPYSL3 upon tumor cells were further investigated. Generating the stable DPYSL3 RNA interference EC cell lines COLO-684 and the stable DPYSL3 overexpression of Ishikawa (mtp53), we examined CCK-8 assay (
CCK-8 assay (A), transwell assay (B), wound healing assay (C) and xenograft mice model assay (D) for investigating the effect of DPYSL3 in EC cells. Data are presented as the mean ± SEM of three individual experiments. * represents the comparison between DPYSL3 knock-down or knock-in and control groups with
To study the anti-proliferative mechanism of DPYSL3 in EC cells, we constructed Stag-Flag-DPYSL3 stably expressing COLO-684 cells, conducted Flag-IP and explored the probable binding protein of DPYSL3 using blue staining. Unlike in empty vector, one interested band near 45 kD was observed and identified as MEK1 using mass spectrometry assay. Moreover, to define the regions of DPYSL3 which are responsible for the interaction with MEK1, we performed a series of deletion analysis on DPYSL3 and conducted reverse IP to validate the interaction between DPYSL3 and MEK. We observed that the N-terminal (2–345) of DPYSL3 was essential for the interaction with MEK (
Co-IP study of the interaction between truncating DPYSL3 protein of amino acids 1–684 (A) or amino acids 1–245 (B) and MEK1/2. The activity of MAPK signaling pathway affected by different truncating DPYSL3 (C). Molecular docking for protein-protein interaction of DPYSL3 and MEK1/2 (D, E). Red is DPYSL3 and green is MEK1/2. The interaction domain between these two proteins is showed as purple (DPYSL3) and yellow (MEK1/2) ribbon.
DPYSL3 is an ectoderm specific gene, mainly expressed during nervous system development in newly born brain (
Recent studies revealed that the DPYSL family were highly expressed in breast and prostate cancers and negatively associated with invasiveness, metastasis, and motility (
The molecular function of DPYSL proteins mainly determine to be able to facilitate Sema3A-mediated morphological changes, such as microtubule dynamics regulation and cytoskeleton redistribution (
In this article, we suggest the role of DPYSL3 in the activity of MAPK/ERK pathway and the effects on tumor malignancy in EC. Our study provides a novel oncotarget for EC tumorigenesis and treatment.
Symbol name | Primer sequence |
---|---|
DPYSL3 for qPCR | F: GGCGGAGATCCACGGTGGA |
R: GGGCCCGTCATACAGTCCACCGT | |
GAPDH for qPCR | F: GAAGGTGAAGGTCGGAGTC |
R: GAAGATGGTGATGGGATTTC | |
DPYSL3 CBS1 | F: ACTCTTCCTTCTAACATGGCAA |
R: GCTGATGCTGATCGGCGCAAC | |
DPYSL3 CBS2 | F: GGGCGGACTGCTAGCTGTCTACA |
R: GTGTCGATTGCTGATCAC | |
DPYSL3 CBS3 | F: AATTTGAGTCGTAACAAAA |
R: TTTATAGCTTACTTCGATCACA | |
DPYSL3 CBS4 | F: AAATTCTGGCGCTCGACTTAC |
R: CCCGGTCGGCATGCTAAAAA | |
DPYSL3 full length | F: AATGCTTTTCAGATTAAACTTA |
R: AATGCTGCTGCTGTACACAT | |
DPYSL3 2-16 deletion | F: GGATGACGATGACAAATACAAGG |
R: GCGTAAAAGGACTGGTCG | |
DPYSL3 17-345 deletion | F: TTCTTGCTCCAGTAGTGTGAA |
R: CCTTATTCCAAGCGGCTTC | |
DPYSL3 346-551 deletion | F: CACGGCTGCGTGATGCTGATGCTACAC |
R: CACGTGCTAGCTGCTAGTGCTAC | |
DPYSL3 552-684 deletion | F: CCCGGCTGCTACTTCGTACACACAAA |
R: AATTTTAGATGTTTACTTTATTCTAA | |
DPYSL3 17-142 deletion | F: CCGGCGGCTTTTCTGCTGCTCGAAAA |
R: CCTTTTGCTGCTGCTTTATTACTTCAAA | |
DPYSL3 143-159 deletion | F: AACCTTCTTTAGGCTGCTGATCGTAA |
R: ACACATGTGATCAGTCGGTTTACCAA | |
DPYSL3 160-245 deletion | F: CCCGGCTGCTTCTTAGTATAAAACCC |
R: GGGGTCGGTCGGCGCGATGCTAC |
Note: All the primers used in this study are listed above.