Fig 2: PAX8, SOX17, and MECOM are candidate MTFs in HGSOC.(A) PAX8, SOX17, and MECOM coincide with SEs in primary ovarian tumors. H3K27ac ChIP-seq data were generated in 12 primary tumors, and data were normalized to counts per million (CPM) mapped reads. (B) SEs were identified in 12 primary HGSOCs; PAX8, SOX17, and MECOM are associated with SEs in most tumors. Stitched enhancers were ranked by H3K27ac ChIP-seq signal. (C) PAX8, SOX17, and MECOM occupy their own and each other’s SEs. Enhancers are indicated as black bars. (D) Proposed core regulatory circuit for HGSOC based on TF co-occupancy. (E) PAX8, SOX17, and MECOM ChIP-seq peaks were ranked by CPM-normalized signal. Strong binding sites for each factor are detected proximal to their own and each other’s gene loci. (F) PAX8, SOX17, and MECOM bind to enhancers of HGSOC clinical biomarkers. (G) Global cobinding of PAX8, SOX17, and MECOM. Each row is a PAX8 ChIP-seq peak. CPM-normalized ChIP-seq reads were plotted for a 4-kb window centered on each binding site. Rows are ordered by decreasing PAX8 signal. (H) PLA performed in Kuramochi cells. Each red dot represents a single interaction. Nuclei were counterstained blue with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 100 μm. Image magnification, ×40. (I) Quantified PLA signal per cell. In (C) and (E) to (G), ChIP-seq data were generated in Kuramochi cells and normalized to CPM mapped reads. ***P < 0.001; Student’s paired t test. Error bars indicate SD of the mean values from three independent experiments (performed with technical triplicates).

Fig 3: Characterization of three HPDL hiPS cells derived from patients’ primary cell lines. (A) Bar plot showing the same or higher expression of OCT4, SOX2, and L-MYC genes in our generated hiPS cells compared with CTRL ones. (B) Bar plot shows the total absence of eOCT4, eSOX2, eLIN28, and eKLF4 episomal gene expression in CTRL and our HPDL hiPS cells compared with tHDFs. (C) Gel image showing the lack of mycoplasma contamination in our patient-derived hiPS cells. (D–F) Electropherograms showing that the parental HDF genotype was maintained in the corresponding hiPS lines. (G1–G3, H1–H3, I1–I3) Representative confocal images with stemness markers b3-tubulin (TUBB3), SOX17, and Brachyury, respectively, in differentiated cells derived from all HPDL hiPS lines. All nuclei were marked with DAPI. Scale bar: 50 μm.

Fig 4: SOX17 is a target gene of miR-194-5p. (A) SOX17 was predicted as the target gene of miR-194-5p by TargetScan. (B) Luciferase reporter assay was performed to verify that SOX17 was a target gene of miR-194-5p. *P<0.05, compared with the UTR-MUT miR-194-5p group. miR-194-5p, microRNA-194-5p; MUT, mutant; NC, negative control; SOX17, SRY-box 17; UTR, untranslated region; WT, wild-type.

Fig 5: PAX8, SOX17, and MECOM are functional dependencies in HGSOC.(A) PAX8, SOX17, and MECOM are selective dependencies and [their gene loci] are commonly amplified in HGSOC tumors (B). (C) Western blot, with quantification, to confirm knockdown of PAX8, SOX17, and MECOM in OVCAR4 cells. (D) PAX8, SOX17, and MECOM coregulation based on normalized quantification of Western blot signal intensities after knockdown. Linewidths denote the percentage of up-regulation (arrows) or down-regulation (flat ends). Solid lines (P < 0.05), dashed lines (not significant) (four independent experiments). (E) Anchorage-independent growth assays. (F) Dose response curves for THZ1, THZ531, and JQ1 treatment. Nonlinear fit curves are shown. Data are representative of three independent experiments. (G) TF expression following a 6-hour drug treatment. (H) PAX8 and SOX17 are among the most sensitive genes in THZ1-treated Kuramochi cells. (I) Ontology analyses for transcripts down-regulated following siPAX8, siSOX17, and siMECOM treatment. IGF, insulin-like growth factor. (J) Retinoblastoma (Rb) pathway genes are among the most significantly down-regulated transcripts following PAX8, SOX17, or MECOM knockdown. (K) GSEA of the top 500 down-regulated genes following PAX8 and SOX17 knockdown, compared to a ranked list of THZ1-responsive genes. (C to G) *P < 0.05, **P < 0.01, and ***P < 0.001; Student’s paired t test; ns, not significant. Error bars indicate one SD of the mean values from three independent experiments (performed with technical triplicates).