Plasmids and cell lines
The p53-specific short hairpin RNA (shRNA) oligonucleotides were designed as described previously [13]. Single-stranded p53 oligonucleotides were annealed and then cloned into BamH1-EcoRV sites of RNAi-Ready pSIREN-RetroQ vector (Takara, Shiga, Japan), according to the manufacturer’s instructions. The p53mt (R248Q) was generated by PCR-based methods using a pCMV-p53wt construct. The human Fibronectin 1 promoter (UCSC genome browser, https://genome.ucsc.edu/) between -2028 and -23 (where +1 represents the transcription start site) was also generated by PCR and was cloned into the pGL3B vector (Promega, Madison, WT, USA). The primer sequences for the PCR reaction used in this study are listed in Table 1. pCMV-p53wt, pGL3B-(-1109/+36) Snail luc, pGL3B-(-899/+47) HNF-1b luc, and pGL3B-(-140/+216) HNF-1b luc were also used as described previously [14-16].
Four OCCCa cell lines, OVISE, ES2, OVTOKO, and TOV-21G were used as described previously [13,16,17], and two OHGSeCa cell lines, OVSAHO and OVCAR-3, were obtained from the National Institute of Biomedical Innovation (Osaka, Japan) and the American Type Culture Collection (Manassas, VA, USA), respectively. p53 shRNA knockdown cells were established using OVISE cells, which have a wild-type p53 gene and abundant expression of endogenous HNF-1b and ARID1A (Supplementary Figure S1), as described previously [13,17]. In addition, spindle-shaped cells were defined as those that showed narrow and elongated phenotypes, along with weak or absent adhesions between cells, as described previously [17].
Antibodies and reagents
Anti-p53, anti-p21waf1, anti-cyclin D1, and anti-bcl2 antibodies were purchased from Dako (Copenhagen, Denmark). Anti-HNF-1b, anti-GSK-3b, anti-Rb, anti-p27kip1, anti-XIAP, anti-bax, and anti-integrin b1 antibodies were obtained from BD Biosciences (San Jose, CA, USA). Anti-ARID1A, anti-cyclin B1, and anti-MDM2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Snail, anti-Akt, anti-phospho(p)Akt Serine473, anti-pGSK-3b Serine9, anti-pRb Serine807/811, anti-cleaved caspase-3, and anti-integrin b3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-fibronectin (FN), anti-E-cadherin, and anti-b-actin antibodies were obtained from Abcam (Cambridge, MA, USA), Takara (Shiga, Japan), and Sigma-Aldrich Chemicals (St. Louis, MO, USA), respectively. Anti-cyclin A2 and anti-integrin b2 antibodies were from Novocastra (Newcastle, UK) and Merck KGaA (Darmstadt, Germany), respectively. FN (catalog number #F2006) and cisplatin (CDDP: #479306) were purchased from Sigma-Aldrich Chemicals.
Transfection
Transfection was carried out using LipofectAMINE PLUS (Invitrogen, Carlsbad, CA, USA) as described previously [14-16]. Luciferase activity was assayed as described previously [14-16].
Reverse transcription (RT)-PCR
cDNA was synthesized from 2 mg of total RNA. Amplification by RT-PCR was carried out in the exponential phase to allow comparison among cDNA synthesized from identical reactions using specific primers (Table 1). Primers for the HNF-1b, Snail, and GAPDH genes were also applied, as described previously [14-16]. The signal intensity was analyzed by ImageJ software version 1.41 (NIH, Bethesda, MD, USA).
For quantitative analysis, real-time RT-PCR was also conducted using a Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). Fluorescent signals were detected using the ABI 7500 Real-time PCR System SDS Software (Applied Biosystems).
Western blot assay
Total cellular proteins were isolated using RIPA buffer [20 mM Tris-HCl (pH7.2), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate]. Aliquots of the proteins were resolved by SDS-PAGE, transferred to membranes, and probed with primary antibodies, coupled with the ECL detection system (Amersham Pharmacia Biotechnology, Tokyo, Japan).
Flow cytometry and Aldefluor assay
Cells were fixed using 70% alcohol and stained with propidium iodide (Sigma) for cell cycle analysis. Aldehyde dehydrogenase 1 (ALDH1) enzyme activity in viable cells was determined using a fluorogenic dye-based Aldefluor assay (Stem Cell Technologies, Grenoble, France) according to the manufacturer’s instructions. The prepared cells were analyzed by flow cytometry using BD FACS Calibur (BD Biosciences) and CellQuest Pro software version 3.3 (BD Biosciences).
Cell Counting Kit-8 assay
The quantitation of viable cell number in proliferation after CDDP treatment was carried out using a Cell Counting Kit-8 (CCK-8; Dojindo Lab, Kumamoto, Japan), according to the manufacturer’s instructions.
Wound healing assay
Cells were seeded into 24-well tissue culture plates, and grown to reach almost total confluence. After a cell monolayer formed, a wound was scratched with a sterile 200-ml tip. The area of the wound was analyzed by ImageJ software version 1.41 (NIH). Cell migration parameters were calculated in pixels as wound closure.
Migration assay
Cell migration was determined using 24-well Transwell chambers with an 8-mm pore size (Corning, NY, USA). The lower chamber was filled with medium containing 10% serum. Cell were suspended in serum-free medium with or without FN and transferred into the upper chamber. After 24 h, the number of cells stained by hematoxylin-eosin (HE) on the bottom surface of the polycarbonate membranes was counted visually using a light microscope.
Apoptotic index
Apoptotic cells were identified in HE-stained sections, according to the criteria of Kerr et al. [18]. A total 10 fields were randomly selected, and the number of apoptotic cells was calculated by counting the mean number of apoptotic figures per high power field (HPF).
NGS assay
Total RNAs were extracted from OV-shp53 and mock cells using the NucleoSpin RNA system (Takara). The concentration and quality of the RNA was verified with the Quantus Fluorometer (Promega) and Agilent 2100 Bioanalyzer, respectively. All the samples showed RIN values over 9. Total RNA (500 ng) was used for RNA library preparation, according to the instructions of the Quant Seq 3’ mRNA-seq library preparation kit FWD from Illumina (Lexogen, Vienna, Austria). The libraries were PCR-amplified for 12 cycles.
Sequencing of the libraries (via single-end 75-bp reads) was conducted on the Illumina NextSeq500 system. All data analyses were conducted using Strand NGS (v3.2, Agilent Technologies). The adapter sequences were removed from the raw reads, and base trimming was performed from the 3’ end of each read to remove bases with quality below Q10 up to a minimum length of 25 bp. Each read was mapped to the reference human genome hg38 with default settings. Expression patterns of transcripts were compared after normalization of DESeq [19] using default settings.
TCGA data analysis
The Cancer Genome Atlas (TCGA) OHGSeCa annotated TP53 gene alteration and mRNA expression data (RNA Seq V2 PSEM) for HNF-1b and ARID1A were extracted from cBioportal for Cancer Genomics (http://www.cbioportal.org/) for 398 OHGSeCa cases.
Clinical cases
A total of 199 cases of OECa, surgically resected at Kitasato University Hospital between 2006 and 2017, were selected from our patient records according to the criteria of the 2014 World Health Organization classification [20]. All patients underwent oophorectomy with or without hysterectomy. None of the patients had received chemotherapy or any other preoperative treatment, while most patients had received Paclitaxel/Carboplatin-based chemotherapy after surgical treatment. Of these, 99 cases including 41 OHGSeCa and 58 OCCCa showed complete resection of the tumors, while 28 cases including 17 OHGSeCa and 11 OCCCa had residual tumors after debulking surgery.
Evaluation of relapse and disease progression was conducted on the basis of radiologic image findings. The tumor cases investigated were comprised of 58 OHGSeCa, 9 ovarian low grade serous carcinoma, 29 ovarian endometroid carcinomas, 71 OCCCa, and 30 ovarian mucinous carcinomas. All tissues were routinely fixed in 10% formalin and processed for embedding in paraffin wax. Approval for this study was given by the Ethics Committee of the Kitasato University School of Medicine (B16-10).
Immunohistochemistry (IHC)
IHC was performed using a combination of the microwave-oven heating and polymer immunocomplex (Envision, Dako) methods, as described previously [14-16].
For evaluation of IHC findings, scoring of nuclear/cytoplasmic immunoreactivity was performed, on the basis of the percentage of immunopositive cells and the immunointensity, with multiplication of values of the two parameters, as described previously [14-16]. In addition, FN/p53 score was generated by multiplication of the values of the two scores.
To evaluate the prognostic significance of FN and p53 expression, the scores were divided into two categories (high and low) with the mean values as the cut-off in each category (Table 2). With regard to p53, cases that were completely negative for p53 immunoreactivity were categorized into the high p53 score group (score = 12), since combining 2 IHC labeling patterns associated with p53 mutations (0% and 60-100% positive cells) correctly identified a mutation in 94% of cases [ 21].
Statistics
Comparative data were analyzed using the Mann-Whitney U-test. Overall survival (OS) was calculated as the time between onset and death or the date of the last follow-up evaluation. PFS was also examined from the onset of treatment until relapse, disease progression, or last follow-up evaluation. OS and PFS were estimated using the Kaplan-Meier methods, and the statistical comparisons were made using the log rank test. Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. The cut-off for statistical significance was set as p < 0.05.