Plasmids and cell lines
FLAG-tagged S100A1 expression plasmid generated by polymerase chain reaction (PCR) was cloned into p3xFLAG-CMV14 vector (Sigma-Aldrich Chemicals, St. Louis, MO, USA). S100A1-specific short hairpin RNA (shRNA) oligonucleotides were designed as described previously . Single-stranded S100A1 oligonucleotides were annealed and then cloned into BamH1-EcoRI sites of RNAi-Ready pSIREN-RetroQ vector (Takara, Shiga, Japan), according to the manufacturer’s instructions. The primer sequences for the PCR reaction used in this study are listed in Table 1.
Eight Em Ca cell lines (Hec1B, Hec6, Hec108, Hec116, Hec155, Hec251, Hec265 and Ishikawa) were used as described previously . S100A1 expression plasmid or empty vector was transfected into Hec6 and Ishikawa cells (which lack endogenous S100A1 expression) and stable overexpressing clones were established. Conversely, we using an shRNA targeting the S100A1 gene [13, 16] to reduce the levels of S100A1 in Hec251 cells, which have relatively high endogenous S100A1 expression (Additional file 3: Figure S3A). These cells are referred to as H251-S100A1-knockdown (KD) in the manuscript.
Antibodies and reagents
Anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma-Aldrich Chemicals. Anti-p21waf1, anti-cyclin D1, anti-p53, anti-BCL2, and anti-Ki-67 antibodies were obtained from Dako (Copenhagen, Denmark). Anti-p27kip1 and anti-BAX antibodies were from BD Biosciences (San Jose, CA, USA). Anti-MDM2 and anti-S100A1 were from Abcam (Cambridge, MA, USA). Anti-cleaved caspase-3 and anti-cleaved poly (ADP-ribose) polymerase 1 (PARP1)(Asp214)(D64E10) were from Cell Signaling (Danvers, MA, USA). Anti-cyclin B1 and anti-cyclin A2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Novocastra (Newcastle, UK), respectively.
Rapamycin, aphidicolin, and nocodazole for synchronization of cells at G1, early S, and G2/M phases, respectively, were obtained from Calbiochem (Cambridge, MA, USA). Adriamycin (ADR) and Nutlin-3A were from Sigma-Aldrich Chemicals.
Reverse transcription (RT)-PCR
cDNA was synthesized from 2 µg 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 GAPDH gene were also used as described previously [13, 16].
Western blot assay and immunoprecipitation
Total cellular proteins were isolated using RIPA buffer [20 mM Tris-HCl (pH 7.2), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate]. Cytoplasmic and nuclear fractions were prepared using ProteoExract Subcellular Proteome Extraction kit (Merck KGaA, Darmstadt, Germany). Proteins were resolved by SDS-PAGE, transferred to membranes, and probed with primary antibodies coupled to the ECL detection system (Amersham Pharmacia Biotechnology, Tokyo, Japan).
For immunoprecipitation, cells overexpressing FLAG-tagged S100A1 were lysed with IP buffer [10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10% NP-40] in the presence of 1 mM CaCl2. Cell lysates were cleared and incubated with anti-FLAG M2, anti-p53 or anti-MDM2 antibodies, followed by incubation with Protein G-Sepharose (Amersham Pharmacia Biotechnology). Western blot assay was subsequently performed with anti-FLAG M2, anti-p53, and anti-MDM2 antibodies.
Cells were fixed using 70% alcohol and stained with propidium iodide (Sigma) for cell cycle analysis. Cells were analyzed by flow cytometry using BD FACS Calibur (BD Biosciences) and CellQuest Pro software version 3.3 (BD Biosciences).
Apoptotic cells were identified in hematoxylin-eosin (HE)-stained sections according to the criteria of Kerr et al. . Five fields were randomly selected, and the number of apoptotic cells was calculated by counting the mean number of apoptotic cells per high power field (HPF).
After transfection of FLAG-tagged S100A1, the cells were incubated with anti-FLAG M2 antibody. FITC- or rhodamine-labeled anti-mouse or rabbit IgG secondaries (Molecular Probes, Leiden, The Netherlands) were used as described previously .
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 scratch wound was made with a sterile 200-µl tip. The area of the wound was analyzed by ImageJ software version 1.41 (NIH). Cell migration was calculated based on the number of pixels occupied by the wound closure compared to control scratches.
Cell migration was determined using 24-well transwell chambers with 8-µm pore size (Corning, NY, USA). The lower chamber was filled with medium containing 10% serum. Cells were suspended in the serum-free upper medium and transferred into the upper chamber. After 48 h, the number of HE-stained cells on the bottom surface of the polycarbonate membranes was counted using a light microscope.
Histological findings were reviewed in hysterectomy specimens of endometrioid-type Em Cas from the case records of Kitasato University Hospital during the period from 2007 to 2020, according to the criteria of the 2014 World Health Organization classification . Each case was also staged according to the 2009 International Federation of Gynecology and Obstetrics (FIGO) staging system and the TNM classification . A total of 189 Em Ca cases, including 109 of grade (G)1, 51 of G2, and 29 of G3 were investigated. The mean age of the patients was 59.1 years (range from 31 to 92), and 113 were post-menopausal. In addition, our cases included 136 subcategorized as clinical FIGO stage I and 40 as stage II-IV, 77 with upper (<1/2) myometrial invasion and 98 with lower (≧1/2) myometrial invasion, as well as 20 that were positive for nodal metastasis and 156 that were negative.
Eighty-nine biopsy specimens of normal endometrial tissues including 32 in the proliferative phase, 41 in the secretory phase (18 early and 23 middle and late), and 16 in the menstrual phase were also investigated. All tissues were routinely fixed in 10% formalin and processed for embedding in paraffin. Approval for this study was given by the Ethics Committee of Kitasato University School of Medicine (B19-144).
IHC was performed using a combination of the microwave oven heating and polymer immunocomplex (Envision, Dako) methods as described previously [15, 16].
For evaluation of IHC findings, scoring of cytoplasmic and/or nuclear immunoreactivity for S100A1, p53, MDM2, p21waf1, and Ki-67 in normal endometrial tissues was performed based on the percentage of immunopositive cells and the immunointensity; the values of these two parameters were multiplied together as described previously [13, 16]. In Em Ca tissues, scoring of S100A1 and p53 immunoreactivities was also carried out in a similar manner. Nuclear immunopositivity for MDM2, p21waf1, and Ki-67 was counted in at least 1000 cells from five randomly selected fields and the labeling indices (LIs) were then calculated as a percentage. In addition, the number of cleaved PARP1-positive cells in five randomly selected fields was used to calculate the mean number of apoptotic figures per HPF.
To examine an association between S100A1 immunoreactivity and several clinicopathological factors, the score was divided into two categories (high and low) with a mean score of 4.5 used as cutoff.
TCGA data analysis
mRNA expression data (RNA Seq V2 PSEM) for the S100 family in 520 cases were extracted from cBioportal for Cancer Genomics (http://www.cbioportal.org/). The data were subcategorized into ‘high’ and ‘low’ groups (scores >0 and 0, respectively) based on the median Z score (= 0) for mRNA expression levels in each category, and then examined for any correlation with overall survival (OS) or progression-free survival (PFS).
Comparative data were analyzed using the Mann-Whitney U-test, and Spearman’s correlation coefficient. The cutoff for statistical significance was set as P < 0.05.