PTEN overexpression and nuclear β-catenin stabilization promote morular differentiation through induction of epithelial–mesenchymal transition and cancer stem cell-like properties in endometrial carcinoma

Plasmids and cell lines

The human PTEN cDNA was kindly provided from Dr Suzy Baker (St. Jude Children’s Hospital) and was subcloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA). The human cyclin D2 (CCND2) promoter (encompassing − 2329 to + 4 bp, where + 1 represents the transcription start site) was amplified by polymerase chain reaction (PCR) and subcloned into the pGL-3B vector (Promega, Madison, WT, USA). A series of 5ʹ-truncated promoter constructs and a cyclin D2 promoter with mutation of a putative TCF4-binding site were also generated by conventional or inversed PCR-based methods. In addition, the human EBP50 promoter encompassing − 1575 to − 70 bp was also subcloned into the pGL-3B vector (Promega) in a similar manner. The identity of all constructs was confirmed by sequencing prior to use. The sequences of PCR primers employed in this study are listed in Additional file 1: Table S1. The pcDNA-β-catenin (deleted S45), pCI-p300, pSG5-HEG0 (estrogen receptor (ER)-α), Glutathione S-transferase (GST)-fusion protein constructs including full length, PDZ1, PDZ2, and EB domains of EBP50, p3xFLAG-CMV14-EBP50, and TOP-reporter constructs were as described previously [15, 16].

Six Em Ca cell lines (Ishikawa, Hec6, Hec50B, Hec59, Hec88 and Hec265) were used as described previously [4]. The PTEN expression plasmid or empty vector was transfected into Hec6 cells (with a lack of endogenous PTEN expression due to the gene deletion) (Additional file 2: Fig. S1), and clones stably overexpressing (H6-PTEN) were established. EBP50-knockout line (H6-EBP-KO) was also generated using Hec6 cells (which have relatively high EBP50 expression). Briefly, guide RNA sequence (gRNA: 5ʹ-TCTATCTTCGCACTTTCCAC-3’) was designed using CRISPRdirect (https://crispr.dbcls.jp). The complementary oligonucleotides for gRNA were annealed and cloned into pSpCas9n(BB)-2A-Puro (PX462) V2.0 (Addgene #62,987). The pSpCas9n(BB)-2A-Puro (PX462) V2.0/gRNA construct was transfected into Hec6 cells and EBP-KO lines were also established. In H6-PTEN cells, spindle-shaped cells were defined as those that showed narrow and elongated phenotypes, along with weak or absent adhesion between cells. At least 200 cells were examined and the percentage with this morphology was reported.

Antibodies and reagents

Antibodies used in this study are shown in Additional file 3: Table S2. Adriamycin (ADR: Catalog No. #D1515) was purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA).

Transfection

Transfection was carried out using LipofectAMINE PLUS (Invitrogen), in duplicate or triplicate, in accordance with the manufacturer’s instructions. All reporter assays were carried out with 24-well plates and 0.4 µg of total plasmids. The pRL-TK plasmid (Promega) was used to normalize for transfection efficiency. Luciferase activity was assayed 24 h after transfection using the Dual-luciferase reporter assay system (Promega).

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 (Additional file 1: Table S1). Primers for the GAPDH gene were also used as described previously [17,18,19]. The intensity of individual signals was measured using 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 and immunoprecipitation assays

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]. 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). For immunoprecipitation, cells were lysed with IP buffer [10 mM Tris–HCl (pH 7.6), 100 mM NaCl, 10% NP-40]. Cell lysates were cleared and incubated with anti-PTEN or anti-EBP50 antibodies, followed by incubation with Protein G-Sepharose (Amersham Pharmacia Biotechnology). Western blot assay was subsequently performed with anti-PTEN and anti-EBP50 antibodies.

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).

Spheroid assay

Cells (× 103) were plated in low cell binding plates (Thermo Fisher Scientific, Yokohama, Japan) in Cancer Stem Cell Premium (ProMab Biotech, Richmond, CA). Uniform spheroids of at least 50 µm in diameter were counted approximately two weeks after plating.

Wound healing assay

Cells were seeded into 24-well tissue culture plates and grown to reach 90–100% confluence. After a cell monolayer formed, a wound was scratched with a sterile 200-µl tip. The area of the wound was also analyzed using ImageJ software version 1.41. Cell migration parameters were calculated in pixels as wound closure.

RNA-seq assay

Total RNAs were extracted from CA-ALK 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 prep kit FWD for 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 with DESeq [20] using default settings.

GST pull-down assay

GST-EBP50-full length, GST-EBP50-PDZ1, GST-EBP50-PDZ2, and GST-EBP50-EB were induced by 1 mM isopropyl-β-D-thiogalactopyranoside and purified with glutathione-sepharose beads. Cell lysates were mixed with purified GST-EBP50-full length, GST-EBP50-PDZ1, GST-EBP50-PDZ2, or GST-EBP50-EB immobilized on the beads. Pull-down assays were performed at 4 °C overnight. The beads were then washed thoroughly with wash buffer [10 mM Tris–HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% Nonidet P-40]. Bound proteins were eluted by boiling in SDS-PAGE loading buffer, separated by SDS-PAGE, and detected by immunoblotting and Coomassie Brilliant Blue staining.

Senescence-associated β-galactosidase (SA-β-gal) assay

Cells were stained for SA-β-gal activity as described previously [17]. At least 200 cells were evaluated for SA-β-gal staining and the labeling indices (LIs) were then calculated as a percentage.

Clinical cases

A total of 102 cases of endometrioid-type Em Cas including 38 of grade (G)1, 33 of G2, and 31 of G3 were selected from the case records of Kitasato University Hospital during the period from 2007 to 2021, according to the criteria of the 2014 World Health Organization classification [21]. Of these, 38 cases of G1 or G2 Em Cas with pre-morular and morular lesions were observed. Pre-morule was defined as small morular lesions composed of less than 20 morular cells. 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 (B20-81).

Immunohistochemistry (IHC)

IHC was performed using a combination of the microwave-oven heating and polymer immunocomplex (Envision, Dako) methods using whole sections. Briefly, after ordinary deparaffinization of 4-µm-thick sections, endogenous peroxidase was blocked by treatment of 0.3% hydrogen peroxide in methanol for 30 min. The microwave-oven heating was carried out with three 5-min cycles in either 10 mM citrate buffer (pH 6.0) or Tris buffer (pH 9.0). Routine IHC staining was then conducted using the polymer immunocomplex method. To assess the immunespecificity of each antibody, either normal mouse or rabbit sera was used as negative control instead of primary antibodies. Assessments of each sample (the scoring of IHC features) were made by three observers (AK, MM, and MS) and then compared.

For evaluation of IHC findings, scoring of cytoplasmic, membranous, or nuclear immunoreactivities in morular, premorular, and the surrounding carcinoma (Sur Ca) components, respectively, was performed on the basis of the percentage of immunopositive cells and the immunointensity with multiplication of the values of the two parameters as described previously [17,18,19]. Nuclear Ki-67 immunopositivity was also counted in at least 200 cells from the three lesions, respectively, and the LIs were then calculated as a percentage.

Immunofluorescence

The slides were heated in 10 mM citrate buffer (pH 6.0) for 3 × 5-min cycles using a microwave oven and then incubated overnight with anti-PTEN, anti-β-catenin, or anti-EBP50 antibodies. Alexa 488 and 570 (Thermo Fisher Scientific, Waltham, MA, USA) were used as secondary antibodies.

Mutation analyses of the PTEN gene

Genomic DNA was extracted from Hec6 cells using a Wizard Genomic DNA Purification kit (Promega) according to the manufacturer’s instructions. Exons 1 to 9 of the PTEN gene were amplified by PCR and the products were subsequently subjected to direct sequencing PCR as described previously [18]. The sequences of primers used in this study are listed in Additional file 1: Table S1.

Methylation analysis of the cyclin D2 promoter

Genomic DNA extracted from cell lines using a Wizard Genomic DNA Purification kit (Promega) was treated by bisulfate using an EZ DNA Methylation-Gold kit (ZYMO Research, Orange, CA, USA). Bisulfate-treated DNA was amplified by PCR using specific primers for the cyclin D2 promoter and the methylation status was analyzed as described previously [22].

RNAscope assay for PTEN and cyclin D2 mRNA in situ hybridization (ISH)

Expression of PTEN and cyclin D2 mRNA was analyzed using an RNAscope assay (Advanced Cell Diagnostics, Hayward, CA, USA) according to manufacturer’s instructions. The hybridization was performed with targeted probes: Hs-PTEN (#408,511), Hs-CCND2 (cyclin D2)(#470,031), positive control probe (#2,010,684), and negative control probe (#310,043) for 2 h at 40 °C. Numbers of intracytoplasmic ISH signals were counted in at least 50 cells and were then expressed as an average number of signals per cell.

Statistics

Comparative data were analyzed using the Mann–Whitney U-test and Spearman’s correlation coefficient. The cut-off for statistical significance was set as p < 0.05.

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