Journal: Cancer cell

Article Title: Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis

doi: 10.1016/j.ccell.2019.12.007

Figure Lengend Snippet: (A) Pie chart of the fraction of circRNAs detected in the indicated number of melanocyte or melanoma STC samples. (B) MDS plot of melanocytes (n=4) and STCs (n=10) defined by circRNA expression. (C) Hierarchically-clustered heat map of circRNAs differentially expressed in melanoma STCs (n=10) vs. melanocytes (n=4) (EdgeR, logFC >1, FDR <0.05). (D) Average correlation coefficients (across samples, Pearson, r) between circular and linear RNAs, grouped by the number of samples in which the circRNA was detected. Mean ± SD per sample group are presented. (E) Cumulative distribution plot of the average circular/linear ratios across samples. (F) Backsplice read counts for CDR1as in melanocytes and individual melanoma STCs. Count or mean count ± SD. (G) FPKM or mean FPKM ± SD of CDR1as from cultured melanocytes (n = 6), and melanoma STCs (n = 13) and cell lines (n = 7). ND = not detected. Red hashed line set to mean FPKM of melanocyte samples. Color coding of cell lines indicating high (red circle), moderate (yellow circle) or low/absent (blue circle) CDR1as expression in subsequently used cell models. (H) Median normalized CDR1as expression (RT-qPCR) in cultured melanocytes (n = 3) and melanoma cell lines (n = 1 per cell line). I, Representative images of single molecule in situ hybridization (smISH) for CDR1as or PPIB (control) in high- and low-expressing melanoma cells. Scale bar is 100 μm. (J) Median normalized expression (log2) of CDR1as by RT-qPCR of melanoma patient samples, grouped by primary (n = 53) and metastatic (n = 52) samples. Primary samples grouped by Breslow thickness (<1 mm or >1 mm). Metastatic samples grouped as in-transit (ITM), lymph node (LN), or distal metastases (DM). (K-M) Median normalized CDR1as expression (RT-qPCR, log2) of primary vs. metastatic samples (K), stage I vs. II samples (L), and ulceration status (primary only) (M). Box plots depict median, 25th and 75th percentile, min-max whiskers. Statistical analyses by two-tailed student’s t test. * p<0.05, ** p<0.01, *** p<0.001 (N-P) Dot plots of median normalized CDR1as expression (log2) from primary melanoma patient samples with Breslow thickness (N), mitoses per mm2 (O), and age at diagnosis (P). Pearson correlation coefficients (r) and p values are indicated. (Q and R) Kaplan-Meier curves of primary melanoma patients stratified by CDR1as expression (Bottom 25% (n = 13) vs. Top 75% (n = 40)) for metastasis-free survival (Q) or overall melanoma-specific survival (MSS) (R). Statistical analyses by Log Rank test.

Article Snippet: CLIP-seq reads for IGF2BP3 were extracted from CLIPdb/POSTAR and mapped in Benchling to the CDR1as sequence.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, In Situ Hybridization, Control, Two Tailed Test, Biomarker Discovery

Journal: Cancer cell

Article Title: Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis

doi: 10.1016/j.ccell.2019.12.007

Figure Lengend Snippet: (A) Representative immunoblot of IGF2BP proteins from Input, IgG, or the indicated IGF2BP RIP samples. (B) Enrichment of the indicated genes by RT-qPCR of IGF2BP RIP. Mean % of input ± SD from replicate experiments (n = 2) are plotted. Statistical comparisons between IGF2BP and IgG RIPs for each gene were performed by Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli, with Q = 1%. * indicate FDR < 0.01. (C) Normalized cell invasion of control (shNTC) or CDR1as-depleted WM278 melanoma cells co-transfected with control (siNTC) or IGF2BP3-targeting siRNA (siIGF2BP3). Data normalized to cell input controls and scaled to the mean of all conditions within experiments. Normalized, scaled means ± SD of replicate experiments (n ≥ 3). Statistical analyses by two-tailed, paired student’s t test. ** p<0.01. (D) Cumulative distribution plot of RIP/Input ratios (log2FC) from IGF2BP3 RIP-seq, with CDR1as and (+) control transcripts indicated. (E) Snapshot of LINC00632/CDR1as locus with tracks from Input and IGF2BP3 RIP-seq samples. (F) Illustration depicting IGF2BP binding motifs, IGF2BP3 CLIP-seq peaks, and miR-7 seed sites on the CDR1as sequence. (G) Venn diagram of genes up- or down-regulated by CDR1as depletion (red circles) overlapped with IGF2BP3-bound genes (blue circle). ES = enrichment score. Statistical analyses by Fisher’s Exact test. (H) Normalized cell invasion of control (shNTC) or CDR1as-depleted WM278 melanoma cells co-transfected with control (siNTC) or siRNA targeting the indicated gene. Data normalized to the mean of all conditions per experiment (n = 2). Scaled means ± SD of independent transfections (n = 8) are plotted. Statistical comparisons by Mann Whitney test between shCDR1as/siNTC group with shCDR1as/siTargets. * p<0.05, ** p<0.01 (I) Representative immunoblot of the indicated proteins in control (shNTC) or CDR1as-depleted (shCDR1as) WM278 cells co-transfected with control (siNTC) or IGF2BP3-targeting siRNA. Band intensity relative to the shNTC/siNTC condition is shown.

Article Snippet: CLIP-seq reads for IGF2BP3 were extracted from CLIPdb/POSTAR and mapped in Benchling to the CDR1as sequence.

Techniques: Western Blot, Quantitative RT-PCR, Control, Transfection, Two Tailed Test, Binding Assay, Sequencing, MANN-WHITNEY

Journal: Cancer cell

Article Title: Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis

doi: 10.1016/j.ccell.2019.12.007

Figure Lengend Snippet: (A) Heat map depicting differential drug sensitivity of the indicated drugs in CDR1asHi (n = 10) vs. CDR1asLo (n = 21) melanoma cells. (B) Area under the curve (AUC) of dose response curves of the indicated agent for CDR1asHi, CDR1asMed, and CDR1asLo groups. (C) Average dependency score (CERES) for GPX4 sgRNA in CDR1asHi, CDR1asMed, and CDR1asLo groups. (D) MITF or AXL expression (RPKM) in CDR1asHi, CDR1asMed, and CDR1asLo groups. (B-D) Box plots in display median, 25th and 75th percentile, and min-max whiskers. Statistical analyses by two-tailed student’s t test comparing CDR1asLo with CDR1asHi groups. ** p<0.01, *** p<0.001, **** p<0.0001 (E) Mean normalized CDR1as expression ± SD (log2) by RT-qPCR in 5 selected melanoma cell lines. (F) Representative immunoblot of AXL and MITF for cells in (E). (G) Representative RSL3 dose response curves of cells in (E). Viability assessed by Cell Titer Glo (RLU) at t = 24 h post-treatment. Mean ± SD of min-max normalized relative light units (RLU) are plotted. Curves fit by log(inhibitor) vs. normalized response with variable slope and IC50s estimated (μm). (H) Individual IC50s (μm) and group means ± SD of replicate dose response curves of (G) (n = 3). Statistical comparisons between IGR1 or 501MEL with WM278, WM1361a, or WM115 by Sidak’s multiple comparisons test. NS = not significant, *** p<0.001.

Article Snippet: CLIP-seq reads for IGF2BP3 were extracted from CLIPdb/POSTAR and mapped in Benchling to the CDR1as sequence.

Techniques: Expressing, Two Tailed Test, Quantitative RT-PCR, Western Blot

Journal: Cancer cell

Article Title: Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis

doi: 10.1016/j.ccell.2019.12.007

Figure Lengend Snippet: (A) Density tracks of H3K27me3 from melanoma STCs/cell lines (Verfaillie et al., 2015) (GSE60666) in the LINC00632/CDR1as locus. (B) Representative immunoblot of H3K27me3 and total Histone H3 of EZH2 inhibitor-treated 501MEL cells. (C) Normalized abundance (%ChIP/Input, qPCR) of the indicated region from IgG, H3, or H3K27me3 ChIP samples isolated from DMSO- or EZH2 inhibitor-treated (GSK126, 2 mM for 10 days) 501MEL cells. Mean ± SD of a representative experiment. (D and E) Relative expression (RT-qPCR) of LINC00632 transcript variants (ENST00000498732, ENST00000602535, and ENST00000370535, left panel) or CDR1as (right panel) from 501 MEL (CDR1asLo) (D) or SK-MEL-28 (CDR1asLo) (E) cells treated with DMSO or GSK126 (2 μM) for the indicated number of days. Data normalized to DMSO-treated control (first time point). Mean ± SD of a representative time course (n ≥ 2 replicate experiments).

Article Snippet: CLIP-seq reads for IGF2BP3 were extracted from CLIPdb/POSTAR and mapped in Benchling to the CDR1as sequence.

Techniques: Western Blot, Isolation, Expressing, Quantitative RT-PCR, Control

Journal: Cancer cell

Article Title: Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis

doi: 10.1016/j.ccell.2019.12.007

Figure Lengend Snippet: (A and B) Relative expression (RT-qPCR) of CDR1as and LINC00632 in WM278 (CDR1asHi) (A) or WM115 (CDR1asHi) (B) cells expressing doxycycline-inducible control (shNTC) or CDR1as-targeting (shA/B) shRNA. Cells harvested 48-72 h post-induction with doxycycline. Mean ± SD of a representative experiment. Statistical analyses by paired student’s t test of replicate experiments (n ≥ 3). (C and D) In vitro cell proliferation of doxycycline-treated WM278 (C) or WM115 (D) cells of (A and B). Cell abundance measured by absorbance at 595 nm from crystal violet staining. Data normalized to day 0. Mean ± SD of a representative experiment. Replicate experiments (n ≥ 2) were performed. (E and F) Normalized cell invasion of WM278 (E) or WM115 (F) cells expressing dox-inducible control (shNTC) or CDR1as-targeting (shA/B) inducible shRNA. (G) Representative fluorescence images of WM278 cell invasion of (E). Scale bars are 300 μm. (H and I) Normalized cell invasion of SK-MEL-28 (CDR1asLo) (H) or 501MEL (CDR1asLo) (I) melanoma cells expressing dox-inducible control (shNTC) or CDR1as-targeting (shA/shB) inducible shRNA. (J and K) Normalized invasion of 501MEL (J) or SK-MEL-147 (CDR1asMod) (K) cells transiently transfected with control (Empty) or CDR1as-overexpression (ZK-SCAN-CDR1as) constructs. (E,F,H-K) Invasion assay data normalized to cell input controls and scaled to the mean of all conditions within experiments. Normalized, scaled means ± SD of replicate experiments (n ≥ 3) are plotted. Statistical comparisons between shA/B and shNTC or ZK-SCAN-CDR1as and Empty vector by two-tailed, paired student’s t test, * p<0.05, ** p<0.01, *** p<0.001. (L) Tumor growth curves of 451Lu (CDR1asMod) xenografts expressing inducible non-targeting (shNTC, n = 11) or CDR1as-targeting (shCDR1as, n = 11) shRNA. (M) Lung metastasis burden at experimental endpoint as assessed by in vivo luciferase imaging (IVIS) of animals in (L). Radiance (photons/s/cm2/sr) from individual animals and mean ± 95% CI are plotted. Statistical analysis by Mann Whitney test. (N) Representative ex vivo fluorescence images of whole lungs of animals in (L). Scale bars are 2 mm.

Article Snippet: CLIP-seq reads for IGF2BP3 were extracted from CLIPdb/POSTAR and mapped in Benchling to the CDR1as sequence.

Techniques: Expressing, Quantitative RT-PCR, Control, shRNA, In Vitro, Staining, Fluorescence, Transfection, Over Expression, Construct, Invasion Assay, Plasmid Preparation, Two Tailed Test, In Vivo, Luciferase, Imaging, MANN-WHITNEY, Ex Vivo

Journal: Cancer cell

Article Title: Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis

doi: 10.1016/j.ccell.2019.12.007

Figure Lengend Snippet: (A) miR-7-5p expression by RT-qPCR from the indicated melanoma cells expressing dox-inducible non-targeting (shNTC) or CDR1as-targeting shRNA (shA or shB). (B) Average median fluorescence intensity (MFI) of melanoma cells stably expressing a perfectly matched (WT) or seed-mutant (MUT) miR-7 GFP reporter and the indicated dox-inducible non-targeting (shNTC) or CDR1as-targeting (shA/shB) shRNA. (C) Relative expression by RT-qPCR of the indicated miR-7 target genes from WM278 (CDR1asHi) cells expressing dox-inducible non-targeting (shNTC) or CDR1as-targeting (shA/shB) shRNA. (A-C) Mean ± SD of replicate experiments (n ≥ 3) are plotted. Statistical analyses by two-tailed, paired student’s t test. NS = not significant, ** p<0.01 (D) Representative immunoblot of the indicated proteins from WM278 cells expressing dox-inducible non-targeting (shNTC) or CDR1as-targeting (shA/shB) shRNA. Band intensity relative to shNTC condition is shown. (E and F) miR-7-5p expression (E) and normalized invasion (F) of dox-inducible control (shNTC) or CDR1as-targeting (shB) WM278 cells co-expressing Cas9 and the indicated miR-7 sgRNA. Data normalized to cell input controls and scaled to the mean of all conditions within experiments. Normalized, scaled means ± SD of replicate experiments (n ≥ 3). Statistical analyses by Sidak’s multiple comparison test. NS = not significant, * p<0.05. (G) Representative immunoblot images of the indicated proteins from the cells of (F). Band intensity relative to the shNTC/sgNTC condition is shown.

Article Snippet: CLIP-seq reads for IGF2BP3 were extracted from CLIPdb/POSTAR and mapped in Benchling to the CDR1as sequence.

Techniques: Expressing, Quantitative RT-PCR, shRNA, Fluorescence, Stable Transfection, Mutagenesis, Two Tailed Test, Western Blot, Control, Comparison

Journal: Cancer cell

Article Title: Epigenetic silencing of CDR1as drives IGF2BP3-mediated melanoma invasion and metastasis

doi: 10.1016/j.ccell.2019.12.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CLIP-seq reads for IGF2BP3 were extracted from CLIPdb/POSTAR and mapped in Benchling to the CDR1as sequence.

Techniques: Produced, Virus, Subcloning, Recombinant, Membrane, Western Blot, Autoradiography, RNA Extraction, Reverse Transcription, SYBR Green Assay, Sample Prep, Chromatin Immunoprecipitation, cDNA Synthesis, TA Cloning, Sequencing, RNAscope, Cell Culture, Control, DNA Methylation Assay, shRNA, Negative Control, Positive Control, Mutagenesis, Software