Journal: NAR Cancer

Article Title: Charting the multilevel molecular response to palbociclib in ER-positive breast cancer

doi: 10.1093/narcan/zcag003

Figure Lengend Snippet: Cell cycle-related mRNA transcripts are downregulated upon Palbo addition. (A) Experimental schematic. HR+/HER2− T-47D BC cells were treated with DMSO (control) or 1 μM Palbo for 24 h before sample collection and subsequent RNA-seq, ATAC-seq, and proteomics analysis. (B) Volcano plot showing DE of genes after Palbo treatment. Blue points represent downregulated genes (Down); red points represent upregulated genes (Up); gray points are not significant (ns). (C) Plot showing GSEA of all genes after Palbo treatment using hallmark gene sets. Genes were ranked by rank = log 2 foldchange × (1 − P -value). A normalized enrichment score (NES) ≥ abs(1.5) and a P -adjusted value ≤ 0.05 were used as the cutoff values for a gene set to be labeled. Note that “E2F Targets” and “G2M Checkpoint” overlap in NES and P -adjusted values. (D) Leading edge plots of two hallmark gene sets shown in 1C. Upregulated genes are enriched for “Estrogen Response Early” while downregulated genes are enriched for “E2F Targets.” (E) Left: Overlap between periodically expressed genes identified by Dominguez et al. (2016) and genes downregulated after Palbo treatment ( P = 1.36e-44, hypergeometric test). Right: Row-scaled gene expression values (fragments per kilobase million; hereafter FPKMs) of these 183 genes across 2 cell divisions.

Article Snippet: RNA libraries were then generated with KAPA messenger RNA (mRNA) HyperPrep Kit (Roche, KK8580).

Techniques: Control, RNA Sequencing, Labeling, Gene Expression

Journal: NAR Cancer

Article Title: Charting the multilevel molecular response to palbociclib in ER-positive breast cancer

doi: 10.1093/narcan/zcag003

Figure Lengend Snippet: CDK4/6 inhibition promotes a global decrease of proliferation-related proteins. (A) Volcano plot of differential proteins detected after MS. Blue points represent downregulated genes (Down); red points represent upregulated genes (Up); gray points are not significant (ns). (B) Western blot validation showing expression of indicated proteins from samples used for MS. (C) Scatter plot showing Palbo-induced gene expression trends at the mRNA (x-axis) and protein (y-axis) levels. Gene directionality was labeled if meeting FDR ≤ 0.05. Pearson’s R = 0.68, P < 2.2e-16. Legend description: Down, negative mRNA and proteomics log 2 foldchange. Up, positive mRNA and proteomics log 2 foldchange. mRNA down, protein up: negative mRNA log 2 foldchange, positive proteomics log 2 foldchange. mRNA up, protein down: positive mRNA log 2 foldchange, negative proteomics log 2 foldchange. ns: not significant. (D) Overlap between significantly (log 2 foldchange ≤ −1, FDR ≤ 0.05) downregulated mRNAs and significantly (log 2 foldchange ≤ −0.6, FDR ≤ 0.05) downregulated proteins ( P = 2.05–163, hypergeometric test). (E) Overlaps between significantly upregulated mRNAs (log 2 foldchange ≥ 1 and FDR ≤ 0.05) and significantly upregulated proteins (log 2 foldchange ≥ 0.6 and FDR ≤ 0.05). (F) Overlaps between alternatively spliced genes and differentially expressed proteins ( P = 0.7405, hypergeometric test). (G) Overlaps between differentially expressed proteins after Palbo treatment and periodic genes identified by Whitfield et al. (2002). (H) Top GO terms enriched in downregulated proteins. The P -value and q-value cutoffs were both set to 0.05 and the ontology “BP” (biological process) was used. The top five enriched terms are shown. (I) Plot showing GSEA of all proteins after Palbo treatment using hallmark gene sets. (J) Leading edge plots of four representative hallmark gene sets. Left: upregulated proteins are enriched for “Oxidative Phosphorylation” and “Estrogen Response Early.” Right: Downregulated proteins are enriched for “E2F Targets” and “Mitotic Spindle”.

Article Snippet: RNA libraries were then generated with KAPA messenger RNA (mRNA) HyperPrep Kit (Roche, KK8580).

Techniques: Inhibition, Western Blot, Biomarker Discovery, Expressing, Gene Expression, Labeling, Phospho-proteomics

Journal: NAR Cancer

Article Title: Charting the multilevel molecular response to palbociclib in ER-positive breast cancer

doi: 10.1093/narcan/zcag003

Figure Lengend Snippet: Palbociclib-response genes are enriched for essential genes. (A) Left: Gene overlap between differentially expressed genes at the mRNA level from Palbo-treated T-47Ds and genes profiled in DepMap across 52 BC cell lines. Right: Heatmap showing gene essentialities of these 745 genes. NA (not available) values are represented by black in the heatmap. Bottom annotation: HER2 and ER status obtained from DepMap annotations for each cell line. Left annotation: Gene regulation of mRNA post-Palbo treatment. Right annotation: Row labels for genes of interest. (B) Left: proportion of “essential-like” genes and “nonessential-like” DE genes across all 52 cell lines. “Essential-like” genes had essentiality scores ≤−1. “Nonessential-like” genes had essentiality scores >−1. Middle and right: Cumulative distribution function plots of essentiality scores for genes downregulated, upregulated, or not significant (ns) genes after Palbo treatment at the mRNA (middle panel) and protein (right panel) levels. The median score for DepMap common essential genes, −1, is shown by a dashed line. Middle panel: P = 1.16e-10 (downreg versus ns), P = 6.54e-04 (upreg versus ns). Right panel: P = 2.24e-07 (downreg versus ns), P = 2.17e-02 (upreg versus ns). P -values calculated by Kolmogorov–Smirnov (KS) test. Red line: upregulated genes, blue line: downregulated genes. (C) CCND1 essentiality scores across for ER+ (green) and TNBC (gray) cell lines. Median essentiality for each group shown by dashed lines. Comparison of mean essentialities: P = 4.58e-06, Student’s t -test.

Article Snippet: RNA libraries were then generated with KAPA messenger RNA (mRNA) HyperPrep Kit (Roche, KK8580).

Techniques: Comparison