Systematic dissection of the mechanisms underlying progesterone receptor downregulation in endometrial cancer.

UNLABELLED
Progesterone, acting through its receptor, PR (progesterone receptor), is the natural inhibitor of uterine endometrial carcinogenesis by inducing differentiation. PR is downregulated in more advanced cases of endometrial cancer, thereby limiting the effectiveness of hormonal therapy. Our objective was to understand and reverse the mechanisms underlying loss of PR expression in order to improve therapeutic outcomes. Using endometrial cancer cell lines and data from The Cancer Genome Atlas, our findings demonstrate that PR expression is downregulated at four distinct levels. In well-differentiated cancers, ligand-induced receptor activation and downregulation are intact. miRNAs mediate fine tuning of PR levels. As differentiation is lost, PR silencing is primarily at the epigenetic level. Initially, recruitment of the polycomb repressor complex 2 to the PR promoter suppresses transcription. Subsequently, DNA methylation prevents PR expression. Appropriate epigenetic modulators reverse these mechanisms. These data provide a rationale for combining epigenetic modulators with progestins as a therapeutic strategy for endometrial cancer.


SIGNIFICANCE
Traditional hormonal therapy for women with endometrial cancer can be molecularly enhanced by combining progestins with epigenetic modulators, thereby increasing progesterone receptor expression and significantly improving treatment efficacy.


Real-time PCR
Total RNA was extracted from cultured cells using the miRvana miRNA Isolation kit (Ambion, Life Technologies). RNA yield and purity was assessed using a NanoDrop Model 1000 spectrophotometer (Thermo Scientific). Total RNA (500 ng) was oligo-dT reverse transcribed with SuperScript III (Invitrogen, Life Technologies). Real-time PCR was performed in triplicate on an Applied Biosystems Model 7900 Genetic Analyzer under standard conditions using the following primer sequences: progesterone receptor PGR (PRA/B)-

Western blotting
Following treatment, cells were solubilized in cold NP-40 cell lysis buffer (150 mM NaCl, 50 mM Tris/ HCl, pH 7.4, 1% NP-40 with a protease and phosphatase inhibitor cocktail from Pierce) and then sonicated to release nuclear proteins as previously described [4]. Lysates were analyzed by Western blotting with specific primary and HRP-conjugated secondary antibodies. For detection of PR protein, a combination of the PRA/B (#3153, Cell Signaling) and PRB (#3157, Cell Signaling) antibodies at a 1:1000 dilution each was used.

Immunostaining
Cells were grown on glass coverslips and treated with indicated reagents. After fixation with 2% paraformaldehyde and permeabilization with 1% Triton X-100, cells were labeled with a PR antibody (#8757, Cell Signaling) as described [5] followed by Alexa Fluor-488 conjugated secondary antibody. Images were visualized by fluorescence microscopy and acquired with an Olympus BX51 camera.

Methylation-specific PCR and sequencing
DNA methylation was assessed by bisulfite sequencing as previously described [6]. Briefly, genomic DNA (gDNA) was prepared from cultured cells using the DNeasy kit according to manufacturer's recommendations (Qiagen). Yield and purity of the gDNA was verified using a NanoDrop Model 1000 spectrophotometer (Thermo Scientific). Bisulfite conversion of 2μg aliquots of gDNA from each cell line was carried out using the EZ DNA Methylation Direct kit according to manufacturer's recommendations (ZYMO Research).
Bisulfite-converted DNA was PCR amplified using primer sequences flanking a CpG island just upstream of the PGR start codon. Primers were designed to specifically amplify bisulfite converted DNA (bisDNA). Primer sequences: PGRbisFOR

Luciferase assay
Ishikawa cells (5x10 5 ) were plated in 6-well plates and transfected with a reporter plasmid, pPRluc (Signosis), that contains 3 progesterone response elements in tandem. Transfections were performed with FuGene 6 (Roche). After 24 hrs, cells were incubated with 20 nM LBH589 or DMSO control for an additional 24 hr. Luciferase activities were measured using a Dual-Luciferase Reporter Assay System (Promega) and a microplate luminometer (Infinite M200, TECAN). Data were normalized to total protein concentration as determined based on the method of Bradford (Bio-Rad). Results are representative of at least 3 independent experiments. All experiments were performed in triplicate.

Colony formation assay
Ishikawa cells (750 cells) were seeded in triplicate in 6 well plates. Cells were treated with DMSO vehicle, 1 μM SAHA or 10 nM LBH589 with or without P4 (100 nM) every other day for 2 weeks. Colonies were stained with crystal violet for image acquisition. Data are presented as the average number of colonies per well. miRNA expression miRNA-specific qPCR assays for miR-96, miR-182, miR-141, miR-129-5p, and miR-375 were carried out using miRNA-specific RT primers and qPCR primer/probe sets (Life technology, Applied Biosystems) on an RNA panel composed of the matched adjacent non-malignant tissue and endometrial endometrioid adenocarcinomas tissue as previously described [7]. These assays were run on an Applied Biosystems Model 7900 Genetic Analyzer and the resulting data again were analyzed using the Applied Biosystems StatMiner software following normalization against the RNU48 endogenous RNA control [7].
Ishikawa H cells were transfected with anti-miR96 inhibitor (Life Technology) using Lipofectamine RNAiMAX (Invitrogen). Total RNA was extracted 48 hours after transfection and miRNA and mRNA expression was measured using q-PCR. Comparisons of miRNA normalized expression values (ΔCt) employed the conventional ΔΔCt fold change method [2,3].

TCGA data analysis
Patient information was downloaded from The Cancer Genome Atlas Data Portal maintained by National Cancer Institute and National Human Genome Research Institute. Gene expression was assayed based on mRNA sequencing conducted on the Illumina platform and was downloaded from NCI's www.impactjournals.com/oncotarget/ Cancer Genomics Hub (CGHub). The calculated expression was for all reads aligning to a particular gene per sample. There were a total of 361 endometrial cancer patients eligible for progesterone receptor gene expression analysis. Patients were divided into four groups: endometrioid grade 1, endometrioid grade 2, endometrioid grade 3 and serous grade 3 which includes cases designated as high grade and mixed histology type. One Way ANOVA was used to detect a significant difference between the groups and the Holm-Sidak method was used for pairwise comparisons. Significance was set as p ≤ 0.05.

Statistical analyses
Student's t-test was used for comparisons of two groups. All pairwise multiple comparisons were performed by one-way ANOVA using the Holm-Sidak method or Bonferroni post-hoc tests with the overall significance level at 0.05 (p ≤ 0.05).