Journal: Science signaling

Article Title: The HDAC3-SMARCA4-miR27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma

doi: 10.1126/scisignal.aau7632

Figure Lengend Snippet: (A) Diagrammatic representation of checkpoint adaptation, a process whereby tumor cells survive chemotherapy and radiation in late phases of the cell cycle. XRT, X-ray telescope radiotherapy; PLK1, polo-like kinase 1; AURKB, aurora kinase B; GSG2, genomic structure of haspin; CDC25B, cell division cycle 25b; CCNB1/2, cyclin B1/2; DS, double-stranded. (B) Basal PAX3:FOXO1 protein expression in murine U23674 aRMS cells transfected with control shRNA (shCtrl), PAX3:FOXO1-targeted shRNA (shYFP), or targeted shRNA plus a lentivirus expressing PAX3:FOXO1 (shYFP + PAX3:FOXO1). Blot is representative of N=3 biological replicates. (C) Viability of the U23674 cells described in (B) exposed to vincristine (VCR) at 4 nM for 24 hours. Graph plotted using GraphPad Prism. Data are mean ± S.D. of N= 3 independent experiments. (D) Box-and-whisker plot showing the tumoristatic efficacy of entinostat (ENT) or vincristine (VCR), alone and in combination, in aRMS mice at day 13 (DMSO vs ENT+VCR). Treatment at a daily dose of 5 mg/kg of ENT by intraperitoneal (IP) injection, VCR at a dose of 1 mg/kg weekly by IP injection, or a combination of both. Data are means ± SEM (N=5 mice per cohort), ***P < 0.001 by log-rank test. (E) Kaplan-Meier plot of the proportion of mice with tumors smaller than 1.2 cubic cm after treatment with ENT and/or VCRat a daily dose of 5 mg/kg of ENT by intraperitoneal (IP) injection, VCR at a dose of 1 mg/kg weekly by IP injection, or a combination of both. Treatment was stopped after day 13 because body weight loss approached 10–15%. Data are means ± SEM (N=5 mice per cohort), *P < 0.05 by log-rank test. In this experiment, treatment was stopped for all mice after day 13 because body weight loss approached 10–15%.

Article Snippet: For analyzing endogenous wild-type FOXO1 expression, recombinant FOXO1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002015","term_id":"1519242198","term_text":"NM_002015"}} NM_002015 ) (Origene) was used.

Techniques: Expressing, Transfection, shRNA, Whisker Assay, Injection

Journal: Science signaling

Article Title: The HDAC3-SMARCA4-miR27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma

doi: 10.1126/scisignal.aau7632

Figure Lengend Snippet: (A-H) Graphical analysis of tumor volume inhibition by either ENT alone or in combination with VCR in 8 different PDX aRMS mouse models (Champions Oncology and Jackson Lab) established from clinical biopsies, recurrent aRMS, or autopsies. Demographic features of these models are in tables S3 and S4; treatment schedules are in table S2. Statistical analyses are given in tables S6–S13. (I) Waterfall plot showing tumor growth inhibition (%) for combination treatment (ENT+VCR) in the 8 models shown in panels (A-H), as labeled. (J) Pharmacodynamic assessment of tumor lysates from J77636 treated with ENT for PAX3:FOXO1 protein expression. Blot shows N=3 biological replicates (N represents tumor lysates from 3 mice per cohort).

Article Snippet: For analyzing endogenous wild-type FOXO1 expression, recombinant FOXO1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002015","term_id":"1519242198","term_text":"NM_002015"}} NM_002015 ) (Origene) was used.

Techniques: Inhibition, Labeling, Expressing

Journal: Science signaling

Article Title: The HDAC3-SMARCA4-miR27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma

doi: 10.1126/scisignal.aau7632

Figure Lengend Snippet: (A and B) PAX3:FOXO1 expression in Rh30 cells transfected with siRNA at 100 nM for 72 hours targeting HDAC1, HDAC2, and/or HDAC3 (A), as well as HDAC10 (B). Blots are representative of N=3 independent experiments. (C-F) Three aRMS samples [U23674 in two replicates (U23674A and U23674B), Rh30, and Rh41] were sequenced after treatment with ENT at 2 μM for 72 hours or DMSO. Featured genes were decreased (log2 ratio of ENT-induced expression divided by control expression > 1) in all samples. Additional expression data for these key targets were curated from previous publications (10, 33) and PAX3:FOXO1 binding data was curated from the literature as indicated (31, 32) or was generated through ChIP-Seq experiments. Four subclasses of ENT-induced gene expression were identified as (C) PAX3:FOXO1 binding with or without HDAC binding, (D) HDAC1, 2, 3 or 11 binding only, (E) indirect targets of PAX3:FOXO1, and (F) otherwise-regulated genes.

Article Snippet: For analyzing endogenous wild-type FOXO1 expression, recombinant FOXO1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002015","term_id":"1519242198","term_text":"NM_002015"}} NM_002015 ) (Origene) was used.

Techniques: Expressing, Transfection, Binding Assay, Generated, ChIP-sequencing

Journal: Science signaling

Article Title: The HDAC3-SMARCA4-miR27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma

doi: 10.1126/scisignal.aau7632

Figure Lengend Snippet: (A) Immunoblots of PAX3:FOXO1, wild-type PAX3, and wildtype FOXO1 in murine C2C12 myoblasts and U23674 aRMS cells upon 72 hours’ treatment with entinostat (ENT; 1 μM) and panobinostat (PAN; 45 nM). Blots are representative of N=3 independent experiments. Note that in the murine aRMS GEM culture U23674, both wild-type PAX3 alleles are homozygously replaced by PAX3:FOXO1, but both FOXO1 alleles are intact (homozygous wild-type). (B-E) Densitometric analysis of (A) PAX3:FOXO1, wild-type PAX3 and wild-type FOXO1expression upon treatment with ENT and PAN. Data are means ± SD, N=3 independent experiments; ***P < 0.001 by two-sided Student’s t test. (F) Western blots of PAX3:FOXO1, wild-type PAX3, and wild-type FOXO1 in HSMM, Rh30 and CF-1 cells upon 72 hours’ treatment with ENT (1μM) or PAN (45 nM). Blots are representative of N=3 biological replicates. In contrast to murine aRMS cultures, PAX3 is heterozygous (wild-type/PAX3:FOXO1) and FOXO1 is heterozygous (wild-type/null) in human aRMS cultures, and both are homozygous wild-type in human HSMM myoblasts. (G-I) Densitometric analysis of PAX3:FOXO1, wild-type PAX3 and wild-type FOXO1 protein expression upon treatment with ENT. Data are means ± SD, N=3 independent experiments; * P < 0.05, ***P < 0.001 by two-sided Student’s t tests. (J) A diagrammatic representation attributing the effect of ENT on PAX3:FOXO1 expression to PAX3 cis-regulatory elements.

Article Snippet: For analyzing endogenous wild-type FOXO1 expression, recombinant FOXO1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002015","term_id":"1519242198","term_text":"NM_002015"}} NM_002015 ) (Origene) was used.

Techniques: Western Blot, Expressing

Journal: Science signaling

Article Title: The HDAC3-SMARCA4-miR27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma

doi: 10.1126/scisignal.aau7632

Figure Lengend Snippet: (A) Differential expression analysis by RNA-seq for genes associated with chromatin-modifying complexes in Rh30 and U23674 cells after treatment for 72 hours with ENT (1 μM) or panobinostat (45 nM) relative to cells treated with DMSO. SMARCA4 average transcripts per million for DMSO-treated cells: 86.86; for ENT-treated cells: 56.15; and for PAN-treated cells: 72.19. (B) SMARCA4 protein abundance in Rh30 and U23674 aRMS cultures treated with ENT or PAN. Blots are representative of N=3 independent experiments. (C) SMARCA4 protein abundance in Rh30 cells transfected with HDAC2- or HDAC3-targeted siRNA (100 nM for 72 hours). Blots are representative of N=3 independent experiments. (D) PAX3:FOXO1 protein abundance in Rh30 cells transfected with SMARCA4-targeted siRNA (100 nM for 72 hours). Blots are representative of N=3 independent experiments. (E) Quantitative PCR (qPCR) analysis of miR-27a expression in Rh30 cells treated with the SMARCA4 bromodomain inhibitor PFI-3 (10 μM for 24 hours). (F) qPCR analysis of miR-485 in Rh30 cells treated with PFI-3. (G and H) qPCR analysis of miR-27a expression in Rh30 and CF-1 cells transfected with SMARCA4-targeted siRNA (100 nM for 72 hours). Data were normalized to the expression of U6snRNA. Gene expression was quantified using the 2^-dCt method. Data are means ± SD, N=3 independent experiments each in triplicate; * P < 0.05, **P < 0.01 by a two-sided Student’s t test. (I) Diagrammatic representation of how ENT and PFI-3 disrupt SMARCA4-mediated interference of miR-27a transcription.

Article Snippet: For analyzing endogenous wild-type FOXO1 expression, recombinant FOXO1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002015","term_id":"1519242198","term_text":"NM_002015"}} NM_002015 ) (Origene) was used.

Techniques: Expressing, RNA Sequencing Assay, Transfection, Real-time Polymerase Chain Reaction

Journal: Science signaling

Article Title: The HDAC3-SMARCA4-miR27a axis promotes expression of the PAX3:FOXO1 fusion oncogene in rhabdomyosarcoma

doi: 10.1126/scisignal.aau7632

Figure Lengend Snippet: (A and B) Western blot of PAX3:FOXO1 protein abundance in Rh30 and CF-1 cells transfected with mimics of miR-27a (10 μM for 72 hours) or a negative control (ctrl). Also shown is blotting in lysates from untransfected control cells (unt). Blots are representative of N=3 independent experiments. (C and D) Light microscopy images of Rh30 and CF-1 cells transfected with mimics of miR-27a. Images are representative of N=3 independent experiments. Scale bar, 50 μM. (E to H) qPCR analysis of miR-27a (E and F) or miR-485p (G and H) expression in Rh30 and CF-1 cells treated for 72 hours with ENT (1 μM) or PAN (45 nM). (I and J) qPCR of miR-27a expression in Rh30 and CF-1 cells transfected with HDAC2- or HDAC3-targeted siRNA (100 nM). Data were normalized to U6snRNA expression. Gene expression was quantified using the 2^-dCt method. Data are means ± SD, N=3 independent experiments each in triplicate.; * P < 0.05, **P < 0.01, and ***P < 0.001 by a two-sided Student’s t tests. (K) Summary of the HDAC3–SMARCA4–miR-27a–PAX3:FOXO1 regulatory circuit targeted by ENT.

Article Snippet: For analyzing endogenous wild-type FOXO1 expression, recombinant FOXO1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002015","term_id":"1519242198","term_text":"NM_002015"}} NM_002015 ) (Origene) was used.

Techniques: Western Blot, Transfection, Negative Control, Light Microscopy, Expressing

Journal: FEBS Open Bio

Article Title: FOXO1 promotes the expression of canonical WNT target genes in examined basal‐like breast and glioblastoma multiforme cancer cells

doi: 10.1002/2211-5463.13696

Figure Lengend Snippet: FOXO1 RNAi‐targeting or disruption impacted WNT target gene expression. (A) FOXO1 and/or beta‐catenin ( CTNNB1 ) RNAi led to reduced LEF1 , TCF7 , and GADD45A gene expression 24 h post‐transfection in U87MG cells by qRT‐PCR using TUBB as the control. (B) FOXO1 RNAi led to reduced LEF , and GADD45A gene expression 18 h post‐transfection in BT549 cells by qRT‐PCR using TUBB as the control. (C, D) FOXO1 disruption by CRISPR Cas9 genome editing in U87MG cells led to reduced LEF1 and TCF7 gene expression measured by qRT‐PCR using TUBB as the control; disruption of FOXO1 produced a truncated protein as assessed by western blot analysis. The disruption mutant is predicted to express the C‐terminal portion of FOXO1 starting in the middle of the DNA‐binding domain. (E) Exogenous FOXO1‐H215R (DNA‐binding‐defective) significantly restored TCF7 gene expression to FOXO1 disruption mutants. * denotes significantly different by Student's t ‐Test compared with the control ( P < 0.05) with SD error bars. Each experiment had three biologically independent replicates.

Article Snippet: The FOXO1 gene was disrupted in U87MG cells using the OriGene CRISPR Cas9 knock‐out kit Cat#: KN400477 (Rockville, MD).

Techniques: Disruption, Expressing, Transfection, Quantitative RT-PCR, CRISPR, Produced, Western Blot, Mutagenesis, Binding Assay