Fig 1: Evaluation of prognosis and druggability of CTCFL-regulated genes in ovarian cancer tumor samples.a Relative expression of the 34 CTCFL-regulated candidate genes in normal and tumor samples (left panel) and their corresponding fold change (right panel). Genes are grouped into DEGs (p-adj < 0.05) or not DEGs (p-adj = 0.05). b Survival analysis with Cox PH model using four survival data: overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFI). c Average Concordance Index (CI) obtained with the gene expression of the 34 candidate genes using the random survival forest (RSF) model. The dashed horizontal line indicates the average CI of random sets of genes for each survival dataset. d Kaplan–Meier plots of MALT1 (top relevant gene found by OS and DSS RSF models) in combination with CTCFL expression. e CTCFL-regulated protein interaction network found in vitro depicting the druggable targets that are differentially expressed in tumor samples versus normal tissue (TCGA vs GTEx). Red and blue nodes indicate genes upregulated and downregulated in tumor vs normal, respectively. Targeting drugs are shown in red boxes, red drug labels indicate drugs currently clinically used for OC treatment.
Fig 2: CTCFL-driven DEGs in OVCAR3 cell line.a Schematic representation of the workflow followed in this study. RNA-seq data were obtained from OVCAR3 cell lines with CTCFL overexpressed and underexpressed from Salgado-Albarran et al.7 and subjected to differential expression analysis. Next, CTCFL BS identification and network enrichment analysis was performed to obtain a candidate set of genes. Finally, the candidate gene list was evaluated in OC-derived tumor samples and normal tissue. b Experimental validation of CTCFL overexpression at the protein level by Western Blot. The upper panel shows the protein abundance obtained by densitometry. BORIS band is 65 kDa. (*) indicate nonspecific bands. c Transcript levels of CTCFL and CTCF in each experimental condition. d Change in gene expression and statistical confidence of CTCFL and CTCF in each experimental condition vs control. e Venn diagram with the number of DEGs found in each and shared between experimental conditions. f Relative gene expression (row z-score of normalized reads) of the 149 DEGs found in both experimental conditions classified by the direction of gene expression change. Gene names in red indicate previously reported CTCFL transcriptional targets7,31,37,89.
Fig 3: BORIS sf6 is involved in sphere forming ability and cancer initiation ability(A) QRT-PCR of CaSki cells transfected with BORIS siRNAs. BORIS mRNA expression in CaSki cells transfected with BORIS-specific siRNA and scrambled siRNA, detected by qRT-PCR analysis. Each value is the mean ± SD of relative quantity (RQ). *P < 0.001. (B) Sphere formation of BORIS-knocked-down cells. Each value is the mean ± SD. *P < 0.001, **P = 0.003. (C) Images of spheres. Magnification is 10 ×, size bar = 100 µm. (D) RT-PCR using specific primers for BORIS subfamilies. (E) Sphere formation of BORIS variants. Cells with overexpression of each of four different BORIS variants, B0, B3, B6 and C7, were established by using aretroviral vector. Sphere formation assays were performed using stable transfectants. Each value is the mean ± SD. (F) Growth curves of tumors derived from CaSki cells transfected with mock and BORIS B0, B3, B6 and C7. Ten, 102 and 103 tumor cells were injected into BALB/C nude mice, respectively. Each value is the mean tumor volume + SE. *P < 0.05. (G) Tumor incidence and estimated frequency of cancer stem cells. The number indicates tumor-initiation incidence in BALB/C nude mice. Cancer stem cell frequency was calculated by Extreme Limiting Dilution Analysis (ELDA) software. CI = confidence interval.
Fig 4: BORIS and CTCF mRNA levels in cell lines.Levels of (A) BORIS and (B) CTCF mRNA were measured by real time PCR in cell lines and testis (provided as a physiological control), and are reported relative to the levels of internal reference transcript TBP. BORIS mRNA was detectable in the breast cell line, MDAMB436, and in the melanoma-derived cell line, MDAMB435, but not in the remaining breast-derived cell lines. CTCF mRNA levels were near or above the levels of TBP mRNA in all cell lines. Testis expressed the lowest CTCF level, near the level of BORIS in this sample (note the differences in scale between the graphs).
Fig 5: BORIS sf 6-specific CTL response can suppress sphere formation(A) Peptide binding assay. Binding affinity was evaluated by comparing mean fluorescence intensity of HLA-A2 expression in the presence of peptide pulsation to mean fluorescence intensity in the absence of the peptide. CMV and influenza peptides were used as positive controls, and GK12 peptide was used as a negative control. (B) ELISPOT assay. BORIS C34_24(9) peptide-specific cytotoxic T cell (CTL) induction was performed and assessed using the interferon (IFN)-? enzyme-linked immunospot (ELISPOT) assay. HLA-A*0201-positive PBMCs were obtained from four healthy donors. Donors A, B and C were HLA-*A0201-positive and donor D was HLA-*A0206-positive. (C) Tetramer assay of BORIS C34_24(9)-specific CTLs. Fluorescence-activated cell sorting (FACS) was performed with PE-conjugated BORIS C34_24(9) peptide/HLA-A*0201 tetramer and anti-CD8-FITC antibody. BORIS C34_24(9) peptide/HLA-A*0201 tetramer-positive cells were directly sorted and a CTL clone was established. (D) Tetramer assay of a BORIS C34_24(9) peptide-specific CLT clone, E1. CTL clone E1 and negative CTL clone were stained by PE-conjugated BORIS C34_24(9) peptide/HLA-A*0201 tetramer and anti-CD8-FITC antibody and analyzed. (E) ELISPOT assay of CTL clone E1. BORIS C34_24(9) peptide specificity of CTL clone E1 and negative clone evaluated by the ELISPOT assay. (F) LDH release cytotoxicity assay. Specific cytotoxicity for peptide-pulsed T2 cells was aexamined (left panel). Influenza peptide-pulsed T2 cells, peptide (-) T2 cells and K562 cells were uses as negative controls. Specific cytotoxicity for CaSki and CaSki/BORIS B6 cells was examined (right panel). K562 cells were used as a negative control. Each value is the mean ± SE. (G) Blocking by anti-HLA-class I antibody. Cytotoxicity of the E1 clone for CaSki cells and CaSki/BORIS B6 cells was examined using anti-HLA class I mAb W6/32 and anti MHC-class II mAb L243. Each value is the mean ± SE. (H and I) Sphere formation in the presence of BORIS C34_24(9)-specific CTL clone. Five × 103 CaSki cells and 5 × 104 E1 CTL clone or CTL negative clone were co-cultured in a 96-well ultra low attachment plate. On co-culture day 8, a microscope photograph was taken (H). Original magnification is × 100. Size bar is 100 µm. The numbers of spheres were counted (I). Each value is the mean ± SE. (J) Tumor growth of CaSki cells in a therapeutic adoptive transfer model. Each value is the mean ± SE. Asterisks indicate statistically significant difference (p < 0.05).
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