Retarded cell division in the zinc finger domain lacking lysine–threonine–serine-transfected MCF-7 cells

<p><b>Copyright information:</b></p><p>Taken from "The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells"</p><p>http://breast-cancer-research.com/content/9/4/R43</p><p>Breast cancer research : BCR 2007;9(4):R43-R43.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC2206716.</p><p></p> A typical result of three individual assays for the carboxy-fluorescein diacetate succinimidyl ester (CFSE) labeling profiles of gated transfectants of MCF-7 cells. The transfectants (CON and zinc finger domain lacking the lysine–threonine–serine insert (ZF - KTS)) and the days post G418 selection (day 1, day 4, day 7 and day 10) are indicated

Accelerated apoptosis in the zinc finger domain lacking lysine–threonine–serine-transfected MCF-7 cells

<p><b>Copyright information:</b></p><p>Taken from "The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells"</p><p>http://breast-cancer-research.com/content/9/4/R43</p><p>Breast cancer research : BCR 2007;9(4):R43-R43.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC2206716.</p><p></p> Dot plots of a representative experiment of flow cytometric analysis for staining of Annexin V–PE (horizontal axis) and 7-amino-actinomycin D (7-AAD) (vertical axis) in nontransfected MCF-7 cells (CON) and transfected MCF-7 cells (Vec, zinc finger domain lacking the lysine–threonine–serine insert (ZF - KTS) or full-length Wilms' tumor 1 suppressor gene (WT1)). Percentages of cell populations in the different quadrants are indicated. Days after G418 selection are shown

Decrease of endogenous WT1 abundance by overexpression of zinc finger domain in breast cancer cells

<p><b>Copyright information:</b></p><p>Taken from "The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells"</p><p>http://breast-cancer-research.com/content/9/4/R43</p><p>Breast cancer research : BCR 2007;9(4):R43-R43.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC2206716.</p><p></p> Northern blot: total RNAs were isolated from the stable transfectants of MCF-7 cells with either pcDNA3 control (CON) or the zinc finger domain lacking the lysine–threonine–serine insert (ZF - KTS) of Wilms' tumor 1 suppressor gene (WT1) expression plasmids as indicated, and were analyzed with WT1 and β-actin probes. Overexpressed WT1–ZF, endogenous WT1 (FL WT1) or β-actin RNA is indicated. Western immunoblot: whole cell extracts were prepared from the stable transfectants of MCF-7 cells or MDA468 cells with either pcDNAcontrol or ZF - KTS expression plasmids, and were analyzed by western blot with either anti-WT1 and β-actin antibodies. Overexpressed WT1–ZF, endogenous WT1 (FL WT1) and β-actin proteins are indicated

Alteration of expression of WT1 target genes in zinc finger domain lacking KTS-transfected MCF-7 cells

<p><b>Copyright information:</b></p><p>Taken from "The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells"</p><p>http://breast-cancer-research.com/content/9/4/R43</p><p>Breast cancer research : BCR 2007;9(4):R43-R43.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC2206716.</p><p></p> Transient and stable transfectants of MCF-7 cells were analyzed for the expression of Wilms' tumor 1 suppressor gene (WT1) target genes or nontarget genes (horizontal axis) with quantitative real-time PCR assays. The relative expression (vertical axis) of these genes in the transfected MCF-7 cells was calculated as compared with the pcDNAcontrol. Results are the average of three experiments. AREG, amphregulin; BASP1, brain acid soluble protein 1; KTS, lysine–threonine–serine; WT1, Wilms' tumor 1 suppressor gene; ZF, zinc finger domain

Inhibition of transcriptional activity of the WT1 promoter by its zinc finger domain

<p><b>Copyright information:</b></p><p>Taken from "The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells"</p><p>http://breast-cancer-research.com/content/9/4/R43</p><p>Breast cancer research : BCR 2007;9(4):R43-R43.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC2206716.</p><p></p> Schematic drawing for luciferase reporter constructs. The predominant transcription start site (+1) is indicated by an arrow. Small solid triangles indicate Wilms' tumor 1 suppressor gene (WT1) binding sites. MCF-7 cells and MDA468 cells were co-transfected with plasmids expressing the WT1 proteins (either pcDNAcontrol (CON), aminoterminal-only construct N-WT1, zinc finger domain lacking or with lysine–threonine–serine (ZF - KTS or ZF + KTS) or full-length WT1 vectors A~D), and either the pGL2 vector control or the differential WT1 promoter-driven luciferase constructs (horizontal axis), respectively. Luciferase activity was normalized with β-galactosidase activity and is expressed in relative luciferase activity as compared with the luciferase vector control (vertical axis). Results are the average of three experiments

Abrogation of WT1-mediated c-transactivation by the zinc finger domain lacking KTS in MCF-7 cells

<p><b>Copyright information:</b></p><p>Taken from "The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells"</p><p>http://breast-cancer-research.com/content/9/4/R43</p><p>Breast cancer research : BCR 2007;9(4):R43-R43.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC2206716.</p><p></p> The MCF-7 cells were transiently co-transfected with the reporter constructs (either pGL2 vector, XNM/Luc or Xmut/Luc), with expression plasmids of Wilms' tumor 1 suppressor gene (WT1) isoforms (either pcDNAvector, A, B, C or D) and with WT1–zinc finger domain (ZF) (either pcDNAcontrol (CON) or ZF lacking the lysine–threonine–serine insert (ZF-KTS)) as well as β-galactosidase expression plasmid, respectively. Relative luciferase activity (vertical axis) is indicated. Histograms show average of three independent experiments

SubID pipeline.

<p>Input containing continuous grouping variable levels and data necessary for calculating an optimization parameter is loaded into SubID (R script), and the population is sorted based on levels of the continuous variable. Next, the population is dichotomized at every percentage from 1 to 99%, and the optimization parameter test is calculated at each cut-off. Permutation and resampling analysis is used to assess FDR. SubID pipeline standard output includes (1) SubID plot, (2) Boxplot depicting levels of continuous variable with outlier status (3) Resampled SubID plot and (4) output plot appropriate for the optimization parameter.</p

Pan-cancer <i>INPP4B</i> prognostic significance.

<p>Standard CoxPH SubID output consisting of (A,E,I) SubID plot, (B,F,J) <i>INPP4B</i> expression boxplot with expression distribution outlier status, and (C,G,K) resampled SubID plot with median (blue) and outlier cut-off values (based on expression distribution; red) for (A-D) kidney clear cell carcinoma, (E-H) bladder urothelial carcinoma, and (I-L) liver hepatocellular carcinoma reveals prognostically significant association between <i>INPP4B</i> expression status and patient survival. Specifically, <i>INPP4B</i>^low is associated with shorter overall survival in (D) kidney, (H) bladder and (L) liver cancers. Furthermore, <i>INPP4B</i> expression status defines three prognostically distinct subgroups within liver cancer.</p

EVI1 regulates <i>INPP4B</i> expression in AML.

<p>(A) <i>INPP4B</i> expression in <i>EVI1</i>^high compared to <i>EVI1</i>^low AML patients both in TCGA-LAML and (B) Verhaak datasets. Data represent mean <i>INPP4B</i> expression in <i>EVI1</i> high vs. low groups; cutoffs were determined using Fisher’s exact optimized SubID. (C) <i>EVI1</i> and <i>INPP4B</i> expression in a panel of AML cell lines. (D,E) <i>EVI1</i> and <i>INPP4B</i> transcript expression in U937 and OCI-AML-3 cells transduced with MSCV-puro-IRES-GFP (PIG) and PIG-EVI1 retroviruses. (F) Illustration of predicted EVI1 binding site 1379bp downstream of the <i>INPP4B</i> transcription start site. (G) Anti-EVI1 ChIP in <i>EVI1</i>^<i>hig</i>h OCI-AML -4 and OCI-AML -6 cells demonstrated enrichment at the +1379bp EVI1 binding site of the <i>INPP4B</i> promoter region.</p

SubID, a non-median dichotomization tool for heterogeneous populations, reveals the pan-cancer significance of <i>INPP4B</i> and its regulation by EVI1 in AML

<div><p>Our previous studies demonstrated that INPP4B, a member of the PI3K/Akt signaling pathway, is overexpressed in a subset of AML patients and is associated with lower response to chemotherapy and shorter survival. <i>INPP4B</i> expression analysis in AML revealed a right skewed frequency distribution with 25% of patients expressing significantly higher levels than the majority. The 75% low/25% high cut-off revealed the prognostic power of <i>INPP4B</i> expression status in AML, which would not have been apparent with a standard median cut-off approach. Our identification of a clinically relevant non-median cut-off for <i>INPP4B</i> indicated a need for a generalizable non-median dichotomization approach to optimally study clinically relevant genes. To address this need, we developed Subgroup Identifier (SubID), a tool which examines the relationship between a continuous variable (e.g. gene expression), and a test parameter (e.g. CoxPH or Fisher’s exact <i>P</i> values). In our study, Fisher’s exact SubID was used to reveal EVI1 as a transcriptional regulator of <i>INPP4B</i> in AML; a finding which was validated <i>in vitro</i>. Next, we used CoxPH SubID to conduct a pan-cancer analysis of INPP4B’s prognostic significance. Our analysis revealed that <i>INPP4B</i>^low is associated with shorter survival in kidney clear cell, liver hepatocellular, and bladder urothelial carcinomas. Conversely, <i>INPP4B</i>^low was shown to be associated with increased survival in pancreatic adenocarcinoma in three independent datasets. Overall, our study describes the development and application of a novel subgroup identification tool used to identify prognostically significant rare subgroups based upon gene expression, and for investigating the association between a gene with skewed frequency distribution and potentially important upstream and downstream genes that relate to the index gene.</p></div