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. 2019 Aug 7;10(1):3554.
doi: 10.1038/s41467-019-11403-6.

ARID1A and PI3-kinase Pathway Mutations in the Endometrium Drive Epithelial Transdifferentiation and Collective Invasion

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Free PMC article

ARID1A and PI3-kinase Pathway Mutations in the Endometrium Drive Epithelial Transdifferentiation and Collective Invasion

Mike R Wilson et al. Nat Commun. .
Free PMC article

Abstract

ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. Here we show that monoallelic loss of ARID1A in the mouse endometrial epithelium is sufficient for vaginal bleeding when combined with PI3K activation. Sorted mutant epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT). We further show that ARID1A is bound to promoters with open chromatin, but ARID1A loss leads to increased promoter chromatin accessibility and the expression of EMT genes. PI3K activation partially rescues the mesenchymal phenotypes driven by ARID1A loss through antagonism of ARID1A target gene expression, resulting in partial EMT and invasion. We propose that ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates, and that coexistent ARID1A and PI3K mutations promote epithelial transdifferentiation and collective invasion. Broadly, our findings support a role for collective epithelial invasion in the spread of abnormal endometrial tissue.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Development of genetic mouse models representing an allelic series of ARID1A mutations in the endometral epithelium. a UCEC endometrioid patient ARID1A alteration status and co-incidence with PIK3CA mutation, taken from TCGA-UCEC dataset. b LacZ expression (blue) is specific to the endometrial epithelium. Sections were counter-stained with nuclear fast red (scale bar = 400 μm). c Diagram of mutant alleles utilized in this study. d PCR genotyping results to detect LtfCre0/+, (Gt)R26Pik3ca*H1047R, Arid1afl, and Arid1aV1068G. e Representative gross images of mice at time of sacrifice due to vaginal bleeding. White arrows indicate tumors. Size of uterine tumor varies within genotype at time of sacrifice. f Weight of semi-dry mouse uterus by genotype. Control (N = 5), LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/+ (N = 14), LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/V1068G (N = 7), LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl (N = 6) (mean ± s.d; * p < 0.05, unpaired t-test, two-tailed). g Survival of mice, based on time until vaginal bleeding. (Gt)R26Pik3ca*H1047R (N = 5), Arid1afl/fl (N = 7), (Gt)R26Pik3ca*H1047R; Arid1afl/+ (N = 17), (Gt)R26Pik3ca*H1047R; Arid1afl/V1068G (N = 7), (Gt)R26Pik3ca*H1047R; Arid1afl/fl (N = 12). Mice succumb to vaginal bleeding (sample image inset) at a median (μ1/2) of 16 weeks (LtfCre0/+; (Gt)R26Pik3ca*H1047R Arid1afl/fl) or 14 weeks (LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/+, and LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/V1068G), without a significant difference between these genotypes. LtfCre0/+ mice harboring Arid1afl/fl or (Gt)R26Pik3ca*H1047R alone did not develop vaginal bleeding. h H&E staining and IHC for ARID1A, P-S6 and KRT8 (N ≥ 2) of the endometrium at 5 × (scale bar = 200 μm) and 20 × (scale bar = 50 μm) magnification, with x20 magnifications representing portion panel to the right surrounded by black box. ARID1A expression is lost in the endometrial epithelium of LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl mice. P-S6 is shown as marker of AKT pathway activation; KRT8 as a marker of endometrial epithelium arrows indicate endometrial epithelium
Fig. 2
Fig. 2
RNA-seq analysis of EPCAM-positive endometrial epithelial cells isolated via magnetic sorting. a Schematic of EPCAM isolation using anti-EPCAM-PE antibody and anti-PE microbeads. b EPCAM is expressed in the endometrial epithelium of a LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl mouse by IHC (N = 3). Arrows indicate endometrial epithelium (scale bar = 100 μm). c IF staining of EPCAM and ARID1A in mouse endometrium (N ≥ 3). Arrows indicate endometrial epithelium (scale bar = 25 μm). d qPCR analysis of Arid1a gene expression of isolated control (N = 3, pooled groups of six mice) and LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl (mutant) (N = 4, single mice) cells (mean ± s.d; **p < 0.01, unpaired t-test, two-tailed). e, f Pathway enrichment analysis on human orthologs of differentially expressed genes between LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl, and control mice (FDR < 0.05; 3481 genes) for mSigDb Hallmark pathways (e) and Gene Ontology (GO) Biological Process terms (f). g GSEA plots showing significance of Mak et al. pan-cancer EMT signature upregulation within LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl compared to control and UCEC ARID1Amut patients compared to ARID1Awt. h Hierarchical clustering of 77 genes within the Mak et al. pan-cancer EMT signature between control and mutant purified endometrium. Genes found in the Hallmark EMT pathway, and CDH1, are identified
Fig. 3
Fig. 3
LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl gene signature correlates with invasive patient gene expression. a Distribution of TCGA-UCEC endometrioid patient tumors relative to ssGSEA score for human orthologs of LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl gene signature. b Clustered comparison of scaled fold-change values for signature genes between LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl vs. control mice and upper vs. lower quartile of UCEC endometrioid patients. EMT genes from Hallmark pathway and Mak and Tong pan-cancer gene signature are identified. c Scatter plot of Hallmark pathway GSEA Normalized Enrichment Scores (NES) from LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl vs. control (human orthologs) and upper quartile of UCEC endometrioid patients vs. lower quartile. d Scatter plot of Hallmark pathway GSEA NES from upper quartile of UCEC endometrioid patients vs. lower quartile and UCEC endometrioid ARID1Amut (frameshift/truncating alterations) vs. ARID1Awt. e Upper quartile ssGSEA-enriched UCEC endometrioid patients present with higher stage disease relative to all patients (p < 0.01, Chi-squared). f Upper quartile ssGSEA-enriched UCEC endometrioid patients have more invasive tumors relative to lower quartile patients (p < 0.05, unpaired Mann–Whitney U, one-tailed). Box-and-whiskers plotted in the style of Tukey without outliers
Fig. 4
Fig. 4
ATAC-seq analysis of differentially accessible chromatin in LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl endometrial epithelium. a ATAC-seq read density heatmap from naive overlapping peaks of control and LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl EPCAM-positive cells, ranked by total intensity. Reads are centered on the middle of the accessible peak ±3 kb. Control (N = 2, pooled groups of six mice) and LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl (N = 2, single mice). b Peak width distributions of control and LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl ATAC-seq peaks, which are significantly different (p < 10−15, unpaired Mann–Whitney U, two-tailed). c Volcano plot for differential accessibility of ATAC-seq peaks between control and LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl cells. Red points represent significant peaks (FDR < 0.20). d Peak width distribution of differentially accessible peaks. e Magnitude distribution of differentially accessible peaks separated by total peaks (gray) and promoter peaks (red, within 3 kb of TSS). f Detailed peak annotation of increasing and decreasing differentially accessible regions for total, non-repetitive and repetitive peaks based on genome annotation. g Enrichment for significant genomic features among differentially accessible peaks, ranked by p-value. Enrichment ratio is calculated by bp of feature in ATAC peak set compared to background genome. h Histogram of all differential ATAC peaks depicting distance to nearest TSS. Percent of peaks found within + /−10, 30, or 100 kb of the TSS are shown. i Histogram of differential ATAC promoter peaks depicting distance to nearest TSS. j mSigDb Hallmark pathway enrichment of genes with differentially accessible promoter peaks. k Differentially accessible promoter peak clustering based on direction and magnitude of change in gene expression and promoter accessibility. Black bars indicate significant differential gene expression by RNA-seq (FDR < 0.05). l Scatter plot depicting the relationship between direction and magnitude of change in accessibility and gene expression for differential promoter peaks. Accessibility and expression were significantly correlated (rs = 0.26, p < 10−9, Spearman). m mSigDb Hallmark pathway enrichment of overlapping differentially accessible promoters and differentially expressed genes
Fig. 5
Fig. 5
ARID1A binding is associated with accessibility and differential gene expression driven by ARID1A loss in human endometrial epithelial cell line. a Western blot of ARID1A expression in siRNA-treated 12Z cells. β-Actin was used as endogenous control. b Annotation of differentially accessible ATAC peaks (FDR < 0.05) from 12Z siARID1A, separated into fractions by directionality and promoter vs. non-promoter. Significant association (p < 10−500, Chi-squared) between increasing accessibility and promoter status. c Annotation of ARID1A ChIP peaks in wild-type 12Z cells. d Peak width distribution of ChIP peaks. e Histogram of all ChIP peaks depicting distance to nearest TSS. Percent of peaks found within + /−10, 30, or 100 kb of the TSS are shown. f Histogram of ChIP promoter peaks depicting distance to nearest TSS. g Enrichment for significant genomic features among ChIP peaks, ranked by p-value. Enrichment ratio is calculated by bp of feature in ChIP peak set compared to background genome. h de novo Motif enrichment of ChIP peaks genome-wide and at promoters. i mSigDb Hallmark pathway enrichment of genes with ChIP promoter peaks. j Read density heatmap of ARID1A ChIP-seq and ATAC-seq (control) at all gene promoters (N = 24,132), ranked by signal intensity for ARID1A ChIP-seq. k Scatter plot depicting correlation between ARID1A binding and chromatin accessibility (rs = 0.312, p < 10–15, Spearman). l Proportional Euler diagram of overlap between ARID1A binding, decreasing and increasing chromatin accessibility at promoters. m Enrichment for LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl gene signature among 12Z siARID1A differentially expressed genes (p < 10−5, hypergeometric enrichment). n Enrichment of ARID1A binding at 12Z siARID1A differentially expressed genes (p < 10−208, hypergeometric enrichment). o Fold-change in gene expression of siARID1A upregulated genes, segregated based on ARID1A promoter-binding status (p= 0.002, unpaired Mann–Whitney U, two-tailed). Box-and-whiskers plotted in the style of Tukey without outliers. p mSigDb Hallmark pathway enrichment of 12Z siARID1A differentially expressed genes (FDR < 0.0001). q Example browser tracks for ARID1A binding profile. Signal is displayed as log likelihood ratio (logLR). Single replicate signal is represented in light green, overlapping signal is represented in dark green. Green bars represent peaks called
Fig. 6
Fig. 6
PIK3CAH1047R antagonizes ARID1A loss-induced mesenchymal phenotypes. a Western blot of ARID1A, β-Actin, AKT, P-AKT, CDH1, SNAI1, SNAI2, and TWIST1 following co-transfection of siNONtg and empty vector (control), siARID1A and empty vector (siARID1A), siNONtg and pPIK3CAH1047R (PIK3CAH1047R), or siARID1A and pPIK3CAH1047R (siARID1A/PIK3CAH1047R). b Proportional Euler diagram displaying differentially expressed genes (FDR < 0.0001) from siARID1A, PIK3CAH1047R, and siARID1A/PIK3CAH1047R relative to control. c mSigDb Hallmark pathway enrichment for siARID1A, PIK3CAH1047R, and siARID1A/PIK3CAH1047R differentially expressed genes. d Enrichment for LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl mouse signature ortholog genes and Mak et al. pan-cancer gene signature within differentially expressed genes from siARID1A, PIK3CAH1047R, and siARID1A/PIK3CAH1047R relative to control. e, f Fold-change values of experimental groups relative to control for genes in the Mak and Tong pan-cancer EMT signature (e) and the Hallmark EMT signature (f), separated based on direction of gene expression change in siARID1A. Statistic represented is paired Mann–Whitney U (two-tailed). Box-and-whiskers plotted in the style of Tukey without outliers. g Intersection between siARID1A differentially expressed genes relative to control and siARID1A/PIK3CAH1047R relative to siARID1A. h Heat map detailing relative expression of intersecting genes (N = 127) (Fig. 6g) in control, siARID1A, PIK3CAH1047R, and siARID1A/PIK3CAH1047R, and ARID1A promoter binding. These genes were enriched for ARID1A promoter binding (p < 10−18, hypergeometric enrichment). i Expression level of intersect genes (Fig. 6g) in siARID1A, PIK3CAH1047R, and siARID1A/PIK3CAH1047R relative to control. Statistic represented is paired Mann–Whitney U (two-tailed). Box-and-whiskers plotted in the style of Tukey without outliers. j mSigDb Hallmark pathway enrichment for intersecting genes (N = 127) (Fig. 6g). k Changes in relative EMT gene expression upon ARID1A loss and PIK3CAH1047R overexpression as measured by qRT-PCR. Data represents three biological replicates
Fig. 7
Fig. 7
ARID1A loss and PIK3CAH1047R promote myometrial invasion in vivo and migration in vitro. a Western blot of ARID1A, β-Actin, AKT, P-AKT, following co-transfection of shNONtg and empty vector (control), shARID1A and empty vector (shARID1A), shNONtg and pPIK3CAH1047R (PIK3CAH1047R) or shARID1A and pPIK3CAH1047R (shARID1A/PIK3CAH1047R). b Invasion assay of 12Z cells with ARID1A loss and PIK3CAH1047R overexpression. Representative images of calcein AM-stained cells are and total invaded cell counts are shown (scale bar = 500 μm). Data represents four biological replicates (mean ± s.d; *p < 0.05, **p < 0.01, ****p < 0.0001, unpaired t-test, two-tailed). c Migration assay of 12Z cells with ARID1A loss and PIK3CAH1047R overexpression. Upper images are representative of cells 24 h following removal of insert (scale bar = 500 μm). Lower images are maximum intensity confocal projections of cells stained with fluorescent phalloidin to label with F-actin (scale bar = 50 μm). Average Migration represents the average difference distance across each migration front from 0 to 24 h. Migrating cell counts represent number of cells in migration area after 24 h. Data represents three biological replicates (mean ± s.d; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t-test, two-tailed). d Myometrial invasion observed in LtfCre0/+; Arid1afl/fl, and LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl. H&E staining and IHC for KRT8 at 3.33–6.66 × (scale bar = 300–600 μm, as stated on figure) and x20 (scale bar = 100 μm) magnification, with x20 magnifications representing portion panel to the right surrounded by yellow box. White arrows indicate invasive endometrial epithelium. Endo, endometrium; Myo, myometrium. e Images of maximum intensity confocal projections of control and LtfCre0/+; (Gt)R26Pik3ca*H1047R; Arid1afl/fl endometrium sections stained with α-smooth muscle actin (α-SMA) (red), KRT8 (green) and counter-stained with DAPI (blue) (N ≥ 3). White arrows indicate invasive endometrial epithelium (scale bar = 50 or 10 μm, as stated on figure). f Diagram representation of EMT-induced invasive endometrial epithelium following ARID1A loss and PIK3CAH1047R mutation

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