Journal: bioRxiv

Article Title: VGLL1-directed TEAD activation drives endocrine therapy resistance in estrogen receptor positive breast cancer

doi: 10.1101/2020.11.29.402842

Figure Lengend Snippet: a, RNA-seq data represented as a volcano plot showing upregulated genes (red, adjusted P < 0.01, log2 (fold change) > 2.5) and downregulated genes (blue, adjusted P < 0.01, log2 (fold change) < −2.5) in FULVR cells compared to MCF7 cells. VGLL1 and VGLL3 expression is strongly increased in FULVR cells. Classical ER target genes (TFF1, PGR and GREB1) are shown as controls for ER signaling downregulation in FULVR cells. b, Normalized gene expression of TEAD coactivators determined by RNA-seq. VGLL1 and VGLL3 are the only TEAD coactivators that show the strongest upregulation in FULVR cells, as well as being up-regulated in tamoxifen-resistant MCF7 (TAMR) cells. Data are presented as box-and-whiskers plots showing the median of two independent biological RNA-seq replicates (whiskers extend to minimum and maximum values). LTED, long term estradiol deprived. RNA-seq data were obtained from ref . c, RT-qPCR for genes in the YAP/TEAD pathway in MCF7 and FULVR cells. Data are presented as mean + s.e.m of three independent experiments. * P <0.05 (Student’s t-test, two-tailed)). d, Heatmap showing the expression of genes in the YAP/TEAD pathway in FULVR cell lines derived from the ER positive breast cancer cell lines MCF7, T47D or ZR-75-1. Upregulation of VGLL1 expression is common to all the FULVR cell lines. The expression values are represented as log2 (fold change) relative to the corresponding parental cell lines and normalized to GAPDH. e, Immunoblotting showing VGLL1 expression in different FULVR cell lines, accompanied by reductions in ER and PR levels. ER, estrogen receptor; PR-A, progesterone receptor isoform A; PR-B, progesterone receptor isoform B. f, Immunohistochemistry of VGLL1 performed in sections from matched breast cancer samples prior to fulvestrant treatment and after fulvestrant treatment and relapse. VGLL1 staining is significantly higher after relapse on fulvestrant treatment, p =0.0098 (one-tailed Wilcoxon signed rank test). Representative images of matched breast cancer tissue pre and post fulvestrant, show increased VGLL1 staining after fulvestrant treatment and relapse. Scale bar, 100μm.

Article Snippet: Formalin fixed, paraffin embedded breast cancer tissue sections (5 μm) from matched pre- and post-fulvestrant patient biopsies were processed for RNA in situ detection using the RNAscope Multiplex Fluorescent Reagent Kit v2 according to the manufacturer’s instructions (Advanced Cell Diagnostics), using RNAscope probe Hs-VGLL1 446731.

Techniques: RNA Sequencing, Expressing, Gene Expression, Quantitative RT-PCR, Two Tailed Test, Derivative Assay, Western Blot, Immunohistochemistry, Staining, One-tailed Test

Journal: EMBO Molecular Medicine

Article Title: Inhibition of DNA methylation promotes breast tumor sensitivity to netrin‐1 interference

doi: 10.15252/emmm.201505945

Figure Lengend Snippet: Bisulfite PCR sequencing (4 CpGs analyzed, region: light gray boxes in Fig A) indicated that DNA methylation of the NTN1 CpG island (CGi) was inversely correlated with its expression. Pearson correlation, P = 0.008, r = −0.55, n = 18. Bisulfite PCR sequencing (3 CpGs analyzed, region: light gray boxes in Fig A) indicated that DNA methylation of the DAPK1 CGi was inversely correlated with its expression. Pearson correlation, P = 0.003, r = −0.66, n = 22. Tissue microarrays (70 paraffin embedded sections) from human breast carcinomas were immuno‐stained with antibodies against DAPK1, UNC5B, and netrin‐1. Samples were classified in quartiles according to the level of netrin‐1 expression. The levels of DAPK1 and UNC5B expression (index constructed from the percentage of sections exhibiting a positive staining) in the first and fourth quartiles of netrin‐1 expressing groups were compared using a chi‐squared test. Representative staining corresponding to low and high levels of expression is shown for each antibody, and expression levels were determined from the percentage of sections exhibiting a positive staining. As a control, staining of the samples using a non‐related isotype antibody was performed. Quantification of the presence or absence of alterations in gene A was associated with the presence or absence of alterations in gene B in human breast tumors which was determined from cBioPortal web site, z ‐score threshold ± 2.2 Individual methylation plots. Red rectangles represent methylated CpG and blue rectangles unmethylated CpG. Mean DNA methylation inhibition of DAPK1 and NTN1 in MDA‐MB‐231 and HMLER cells upon DAC treatment.

Article Snippet: Tissue microarrays of paraffin embedded breast tumor sections were obtained from Super Bio Chips (Cliniscience, Nanterre, France).

Techniques: Sequencing, DNA Methylation Assay, Expressing, Staining, Construct, Methylation, Inhibition