Background
As one of the most common malignant cancers in gynecological malignancies, ovarian cancer (OV) is the highest leading cause of marked morbidity and cancer-associated mortality worldwide [
1]. Despite recent progress in early diagnosis, chemotherapy, radiotherapy, and surgical resection, most OV patients undergo recurrence or develop resistance to current treatment [
2]. Against this background, it is imperative that a better understanding of the pathological process of OV be reached to facilitate the discovery of novel biomarkers for the successful prevention, diagnosis, and treatment of OV.
There is mounting evidence that Notch can enhance the proliferation and differentiation of ovary stem/progenitor cells [
3‐
6]. For example, the abnormal activation of Notch signaling and its essential roles in cancer development have been demonstrated in multiple types of cancer, including OV [
6‐
10]. Some studies have documented 5 Notch ligands (Delta-like 1, 3, 4, and Jagged 1 and 2) and 4 Notch receptors (Notch 1–4) in the highly conserved Notch signaling pathway of mammals [
11]. In addition, binding of the ligand to the receptor initiates gamma-secretase complex cleavage Notch receptor at the cell membrane, thereby releasing the intracellular signaling fragment of the Notch receptor, known as Notch receptor intracellular domain (NICD). Following this activity is the translocation of the released NICD to the nucleus and assemble of a transactivation complex with the DNA binding protein CSL and coactivator MAML, which could induce the transcription of target genes, such as those in the hairy and enhancer of split (Hes) and Hes related with YRPW motif (hey) -related gene family. The above findings are testimony to the potential effectiveness of targeting Notch signaling as a treatment of OV.
Histone methylation is an essential pattern of epigenetic modification that controls gene expression. The methyltransferases and demethylases of histone lysine mediate the balance of histone methylation, serving critical functions in homeostasis, development, physiology, cancer, and other diseases [
12,
13]. Accumulation of evidence indicated that the aberrant methylation of H3K4 induced multiple tumors development [
14‐
17]. SETD1A, an H3K4 methyltransferase, plays essential roles in multiple types of cancer progression including gastric cancer, colorectal cancer, and breast cancer [
18‐
23], and liver cancer sorafenib resistance [
24]. However, the detailed functions of SETD1A in OV development have not been determined and more studies are required to clarify the potential mechanism.
In this work, we identified the mechanism by which SETD1A enhances Notch signaling and OV tumorigenesis. Our findings add to the functions of SETD1A in OV development and helped to identify a potential target for OV treatment.
Methods
Antibodies and reagents
Antibodies against SETD1A (A300-289 A) was purchased from Bethyl (Montgomery, TX, US). Antibodies against H3K4me3 (ab8580), Hes1 (ab108937), Hey1 (ab22614), Hey2 (ab167280), Heyl (ab26138), Notch1 (ab27526), Ki-67 (ab16667), β-actin (ab8226), and Rabbit IgG-Isotype Control (ab172730) were obtained from Abcam (Cambridge, UK).
Cell Counting Kit (CCK)-8, G418, puromycin, and Trizol were purchased from Sigma-Aldrich (St. Louis, MO, US). Dulbecco’s modified Eagle’s medium (DMEM), RPMI1640, penicillin-streptomycin (P/S), and fetal bovine serum (FBS) were purchased from Gibco (Woodland, CA, USA). RIPA buffer and BCA Kit were purchased from Beyotime Biotechnology (Shanghai, China). Protease inhibitor cocktail was purchased from Roche (Basel, Switzerland). HiScript Q RT SuperMix was obtained from Vazyme (Nanjing, China). Lipofectamine 2000 and SYBR were obtained from Invitrogen (Carlsbad, CA, US). Polyvinylidene difluoride membrane was purchased from Millipore (Bedford, MA, US). A dual-luciferase assay system was obtained from Promega (Madison, WI, US). SimpleChIP® Enzymatic Chromatin IP Kit was obtained from Cell Signaling Technology (Danvers, MA, United States).
Patients and specimens
Clinical specimens from 21 patients with OV were taken from the tumor and adjacent normal ovarian tissues. All the patients who underwent radical ovarian cancer resection had never received radiotherapy or chemotherapy before, and the clinicopathological characteristics were showed in the Table
1. The human studies were approved by the Translational Medical Independent Ethics Committee of Shanghai Ninth People’s Hospital (Shanghai, China). The study methodologies conformed to the standards set by the Declaration of Helsinki.
Table 1
Association between SETD1A expression and clinicopathological characteristics of primary epithelial ovarian cancer in human
Age | | | |
0.018
|
0.893
|
≥ 60 |
15
|
7
|
8
| | |
< 60 |
6
|
3
|
3
| | |
Pathologicl Characteristic
| | | |
0.4
|
0.527
|
Serous carcinoma |
16
|
4
|
12
| | |
Non-serous carcinoma |
5
|
2
|
3
| | |
Differentiation degree
| | | |
3
|
0.083
|
High |
3
|
1
|
2
| | |
Moderate |
8
|
2
|
6
| | |
Low |
10
|
3
|
7
| | |
FIGO stage
| | | |
3.033
|
0.219
|
I
|
3
|
2
|
1
| | |
II
|
6
|
2
|
4
| | |
III-IV
|
12
|
2
|
10
| | |
The expression of SETD1A, clinical survival analysis, and KEGG pathway of ovarian cancer patients
The expression of SETD1A in OV patients and adjacent normal ovarian tissues from Gene Expression Omnibus (GEO) was analyzed by GEO2R. The survival rates of OV patients from GEO and the cancer genome atlas (TCGA) were analyzed by KM plotter: Kaplan-Meier Plotter (
http://kmplot.com/analysis/index.php?p=service&cancer=ovar)[
25]. The SETD1A low or high expression was divided by the best separation of survival. The differently changed genes associated with SETD1A and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed by LinkedOmics (
http://www.linkedomics.org/admin.php) [
26‐
29].
Cell lines and stable cell lines
Human ovarian epithelial cell ISOE80, OV cell lines SKOV3, Caov3, RMGI, OVCAR5 and OVCAR8, and human embryonic kidney 293T (HEK293T) cells were obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). All cell lines were maintained at 37 ℃ in RPMI1640 or DMEM supplemented with 10% FBS and 1% P/S.
Recombinant retroviruses expressing the pBABE vector and SETD1A cDNA were generated according to the manufacturer’s operating procedures. The retroviruses were utilized to infect RMGI and OVCAR5 cells and then stable cell lines with 1 µg/ml puromycin were selected. Recombinant lentiviruses expressing the pll3.7 vector and SETD1A Knockdown were generated according to the manufacturer’s operating procedures. The sequences of SETD1A shRNA are: 5’-GACAACAACGAATGAAATATT-3’ and 5’-CAACGACTCAAAGTATATATT-3’. The lentiviruses were utilized to infect SKOV3 and Caov3 cells, and then stable single clones with 1 µg/ml puromycin were selected.
Real-time quantitative polymerase chain reaction (RT-qPCR)
In compliance with the manufacturer’s operation procedures, Trizol was employed to isolate total RNA, and HiScript Q RT SuperMix was utilized for cDNA synthesis of 1 µg total RNA. The synthesized cDNAs were selected for RT-qPCR using SYBR. The primers of Notch target genes in the current study were listed as follows: Notch1: Forward 5’-GAGGCGTGGCAGACTATGC-3’, Reverse 5’- CTTGTACTCCGTCAGCGTGA-3’; Hes1: Forward 5’-TCAACACGACACCGGATAAAC-3’, Reverse 5’-GCCGCGAGCTATCTTTCTTCA-3’; Hey1: Forward 5’-ATCTGCTAAGCTAGAAAAAGCCG-3’, Reverse 5’-GTGCGCGTCAAAGTAACCT-3’; Hey2: Forward 5’-AAGGCGTCGGGATCGGATAA-3’, Reverse 5’-AGAGCGTGTGCGTCAAAGTAG-3’; Heyl: Forward 5’-GGCTGCTTACGTGGCTGTT-3’, Reverse 5’-GACCCAGGAGTGGTAGAGCAT-3’; β-actin: Forward 5’- GGAGCGAGATCCCTCCAAAAT-3’, Reverse 5’- GGCTGTTGTCATACTTCTCATGG-3’.
Western blot
The total protein utilized for Western blot was extracted with RIPA buffer supplemented with a protease inhibitor cocktail. The protein concentration was quantified using the BCA Kit. The Western blot system was established using a Bio-Rad Bis-Tris Gel System (Bio-Rad, Hercules, CA) following the manufacturer’s operating procedures. The primary antibodies, which had been prepared in TBST with 3% BSA at a 1:1000 dilution, were utilized for incubation with the membrane at 4 °C overnight. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies at a 1:10000 dilution at room temperature for 1 h. After rinsing, the signal in the PVDF membrane which carried blots and antibodies were captured using the Bio-Rad ChemiDoc XRS system (Bio-Rad, Hercules, CA).
Cell viability assay
Cells were seeded in 96-well plates and cultured for a different time. CCK-8 was added into cells for 1 h, and then the OD values were recorded at 450 nm.
Cell numbers assay
For cell numbers counting, cells were seeded in 6-well plates and cultured for a different time, and then harvested. Each 20 µl of cells were mixed together with 20 µl of 0.4% Trypan Blue and maintained for 5 min at room temperature. Cells were counted.
Wound healing assay
Cells were seeded into 12-well plates until the cells reached 95% confluency. The adherent cells were then scratched with a pipette tip (10 µl), and the detached cells were washed gently with PBS. Subsequently, the cells were cultured in a serum-free medium for 48 h. The images at 0 and 48 h were captured with × 100 magnification to evaluate wound healing.
Cell migration and invasion
A modified two-chamber transwell migration and invasion assay was performed to measure cell migration and invasion. Cells that were suspended in 200 µL of medium without serum were plated on the upper chamber without (migration) or with (invasion) gel of 24-well transwell chamber, while 700 µL of medium with serum was thrown into the lower chamber. After being maintained for 48 h, cells were fixed with methanol and the non-traversed cells on the upper surface of the filter were removed. In comparison, the traversed cells on the lower surface of the filter were subjected to staining with 0.1% crystal violet and then counted.
Dual-luciferase activity assay
The Hes1 promoter-reporter was cloned into the pGL3 report vector. Lipofectamine 2000 was used to co-transfect SETD1A and NICD with the reporter constructs. The dual-luciferase assay system was employed to measure the reporter activity according to the manufacturer’s operating procedures.
Co-IP assay
Cells were harvested and lysed in IP lysis buffer (150 mM NaCl, 30 mM Tris, 1% Triton X-100, 0.5 mM EDTA, 100 µM orthovanadate, 10 mM NaF, 200 µM PMSF, 10% glycerol, pH 7.5) supplemented with cocktail. The lysates were incubated with SETD1A or Notch antibody with protein A/G agarose overnight at 4 °C with agitation. The complexes were precipitated and washed.
ChIP assay
ChIP assay was performed using SimpleChIP® Enzymatic Chromatin IP Kit under the manufacturer’s operating procedures. The following primers were utilized to amplify the Hes1 promoter DNA fragment: Forward 5′-CAGACCTTGTGCCTGGCG-3′, and Reverse 5′-TGTGATCCCTAGGCCCTG-3′.
Tumor xenografts
A total of 5 × 106 control or SETD1A knockdown SKOV3 cells were subcutaneously injected into nude mice. Each group has 6 mice. The tumor size of mice was monitored every other 2 days using a vernier caliper from the 7th day after injection. The tumor size was calculated according to the formula: size = 0.52 x length x width2. All animal experiments were according to the ethic regulations and approved by the Translational Medical Independent Ethics Committee of Shanghai Ninth People’s Hospital (Shanghai, China).
Immunohistochemistry
Slides were deparaffinized and then subjected to antigen retrieval and H2O2 treatment followed by blocking in 3%BSA for 30 min. Next, the samples were stained with Ki-67 antibody (1:200) overnight at 4 °C. On the next day, the sections were incubated with alkaline phosphatase-conjugated secondary antibody incubation for 1 h at room temperature. The signal was visualized using NBT/BCIP reagent.
Statistical analysis
Means ± standard deviation (SD) was presented as indicators of the results. To compare differences between different groups, the nonparametric Mann-Whitney U test, and unpaired t-test were conducted with the GraphPad Prism software (version 8; GraphPad Software, Inc., La Jolla, CA, USA). p < 0.05 was a statistically significant difference.
Discussion
There is increasing evidence that the abnormal expression and activation of SETD1A promote cancer progression including gastric cancer, colorectal cancer, and breast cancer [
18‐
23]. However, the detailed functions and mechanism of SETD1A in OV development have remained unclear. In this study, the critical role of SETD1A in OV development was demonstrated as follows: (1) SETD1A was overexpressed in OV patients and indicated poor prognosis; (2) SETD1A significantly augmented OV cell proliferation, migration, invasion, and tumorigenesis; (3) SETD1A acted a coactivator to enhance Notch signaling (Fig.
8E).
The classical activation of Notch signaling should be that Notch first binds with its ligand, gamma-secretase complex cleavage Notch, and then, the activated form of Notch translocates to the nucleus to generate a transactivation complex with CSL and MAML, thereby initiating the transcription of Notch target genes. However, what controls the transactivation of the NICD–CSL–MAML complex is still unclear. Increasing evidence has shown that the NICD–CSL–MAML transcriptional complex could interact with several nuclear factors, including p300, GCN5, and PCAF, and then mediate Notch signaling [
30,
31]. In addition, the Notch1 nuclear interactome exhibited critical regulators that initiated Notch signaling through demethylating modifications at the promoter of Notch target genes, highlighting the important role of histone modificator as a coactivator in the mediation of Notch signaling [
32]. In the current study, we found that the histone methyltransferase SETD1A, which methylates H3K4me/3, could augment Notch signaling (Fig.
6). To our best knowledge, this is the very first report that SETD1A could serve as a critical coactivator for Notch signaling.
Notch signaling is implicated not only in OV progression, but also in ovary development and homeostasis, which mediates female reproduction, directly influences fertility in women [
33]. Notch signaling is activated in the adult and neonatal mouse ovaries, and mouse oocytes from entering meiosis are arrested when Notch signaling is inhibited [
33,
34]. Sustained activation of Notch in dedifferentiated parietal cells eventually augmented cell proliferation, and inhibition of Notch signaling reduced OV cell proliferation, migration and invasion, and drug resistance of OV cells [
35‐
38]. These reports suggest that although targeting Notch signaling is a reasonable treatment for OV, directly reducing Notch signaling may weaken the differentiation of stem/progenitor cells, leading to serious side effects. Therefore, there is an urgent need for a strategy that only inhibits Notch signaling in OV cells without affecting healthy ovary cells. Indeed, our research only focused on the role of SETD1A in OV cells. Additional research is required to further investigate the effect of SETD1A on ovary cells.
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