Journal: Nature Communications

Article Title: Epithelial tumor suppressor ELF3 is a lineage-specific amplified oncogene in lung adenocarcinoma

doi: 10.1038/s41467-019-13295-y

Figure Lengend Snippet: ELF3 is located within a region of focal amplification in lung adenocarcinoma. a Comparison of significantly focally amplified regions on chromosome 1q in LUAD and LUSC. b Comparison of ELF3 expression between LUAD (red) and LUSC (green) in TCGA ( n = 1017), Samsung Medical Centre ( n = 138, GSE8894), and Duke University data sets ( n = 111, GSE3141) by Mann–Whitney U test. c Box and whiskers plots of log 2 ELF3 expression in 83 paired cases of non-malignant lung (blue) and lung adenocarcinoma (red) in the BCCA cohort (Wilcoxon sign-rank test), and unpaired cases of 58 non-malignant lung and 513 lung adenocarcinoma samples in the TCGA cohort (Mann–Whitney U test). d Mean log 2 ELF3 expression in non-malignant lung (blue) and in LUAD (red) grouped by purity estimate in the TCGA cohort . e Distribution of ELF3 expression fold change in LUAD compared with paired non-malignant lung in the BCCA and TCGA cohort. f Representative ELF3 immunohistochemistry images from a tissue microarray of LUAD (left panel) and LUSC (right panel) (scale bar = 100 µm). g Box and whiskers plots of ELF3 immunohistochemistry (IHC) score as determined by pathologist review ( n = 236, Mann–Whitney U test). Score was calculated by multiplying the percent of positive cells by the stain intensity (+1, +2, +3). In all box and whisker plots, center line represents the median, box bounds indicate the 25th and 75th percentiles, and whiskers extend from minimum to maximum. LUAD = lung adenocarcinoma, LUSC = lung squamous cell carcinoma, TMA = tissue microarray.

Article Snippet: Relative human cell-specific expression of ELF3 (Hs00963882_g1 – no cross reactivity with mouse) and NOTCH3 (Hs01128537_m1 – no cross reactivity with mouse) was quantified by qRT-PCR (TaqMan—Applied Biosystems) using mouse Actb (Mm04394036_g1) and human Actb (Hs99999903_m1) as endogenous controls.

Techniques: Amplification, Comparison, Expressing, MANN-WHITNEY, Immunohistochemistry, Microarray, Staining, Whisker Assay

Journal: Nature Communications

Article Title: Epithelial tumor suppressor ELF3 is a lineage-specific amplified oncogene in lung adenocarcinoma

doi: 10.1038/s41467-019-13295-y

Figure Lengend Snippet: Genetic and epigenetic alterations at the ELF3 locus. a Case-by-case representation of ELF3 locus deregulation in the BCCA data set ( n = 83 pairs) by genetic and epigenetic events. The frequencies of copy number gain or amplification, and promoter hypomethylation are indicated, as well as the cumulative frequency of ELF3 DNA-level alterations in the BCCA data set. These frequencies are also summarized to the right of the plot for the TCGA ( n = 420) and TCGA-60 ( n = 252) data sets (TCGA cases with ≥ 60% tumor cellularity, see Methods). b Scatter plot of ELF3 promoter methylation ( x axis) and log 2 ELF3 expression ( y axis) across 452 LUAD (gray) and 21 non-malignant lung (blue) samples (Spearman’s correlation). c Log 2 ELF3 expression as a function of DNA copy number alteration in LUAD ( n = 420). ELF3 expression is increased with activating events (Mann–Whitney U test). d Comparison of log 2 ELF3 expression between LUAD with gain and/or promoter hypomethylation (blue) compared with those without (black) ( n = 420, Mann–Whitney U test). In all box and whisker plots, center line represents the median, box bounds indicate the 25th and 75th percentiles, and whiskers extend from minimum to maximum. LUAD = lung adenocarcinoma, AMP = amplification.

Article Snippet: Relative human cell-specific expression of ELF3 (Hs00963882_g1 – no cross reactivity with mouse) and NOTCH3 (Hs01128537_m1 – no cross reactivity with mouse) was quantified by qRT-PCR (TaqMan—Applied Biosystems) using mouse Actb (Mm04394036_g1) and human Actb (Hs99999903_m1) as endogenous controls.

Techniques: Amplification, Methylation, Expressing, MANN-WHITNEY, Comparison, Whisker Assay

Journal: Nature Communications

Article Title: Epithelial tumor suppressor ELF3 is a lineage-specific amplified oncogene in lung adenocarcinoma

doi: 10.1038/s41467-019-13295-y

Figure Lengend Snippet: High ELF3 expression is not dependent on the molecular subtype of lung adenocarcinoma. a Comparison of log 2 ELF3 expression and ELF3 immunohistochemistry (IHC) score between cases with or without driver mutations in EGFR and KRAS (BCCA n = 83, TCGA n = 484, Tissue Microarray n = 161, Mann–Whitney U test). In all box and whisker plots, center line represents the median, box bounds indicate the 25th and 75th percentiles, and whiskers extend from minimum to maximum. b Case-by-case representation of clinical features (upper panel) and genomic alterations (lower panel) across 420 LUAD from the TCGA data set dichotomized into those with and without DNA-level alterations at the ELF3 locus. Mutations and amplifications in prominent oncogenes and mutations in tumor suppressor genes are displayed.

Article Snippet: Relative human cell-specific expression of ELF3 (Hs00963882_g1 – no cross reactivity with mouse) and NOTCH3 (Hs01128537_m1 – no cross reactivity with mouse) was quantified by qRT-PCR (TaqMan—Applied Biosystems) using mouse Actb (Mm04394036_g1) and human Actb (Hs99999903_m1) as endogenous controls.

Techniques: Expressing, Comparison, Immunohistochemistry, Microarray, MANN-WHITNEY, Whisker Assay

Journal: Nature Communications

Article Title: Epithelial tumor suppressor ELF3 is a lineage-specific amplified oncogene in lung adenocarcinoma

doi: 10.1038/s41467-019-13295-y

Figure Lengend Snippet: Manipulation of ELF3 expression regulates oncogenic phenotypes. Histogram summarizing the effect of shRNA-mediated ELF3 inhibition on a , b soft agar colony formation and c cell proliferation in lung adenocarcinoma cell lines, and d the effect of forced ELF3 overexpression (OE) on cell proliferation in HBEC-KT cell lines with and without p53 knockdown (HBEC-KT53) and/or induction of KRAS (HBEC-KTR, HBEC-KTR53). e Tumor growth of isogenic HCC827 shELF3 and control cells in NOD-SCID mice ( n = 12). f Quantification of shELF3 and control vector DNA copy number in endpoint tumors compared with input material. g Putative model of clonal drift throughout xenograft tumor growth. At Day 0 we assume a mixed population of cells with low (blue), medium (pink), and high (orange) vector copy numbers (CN) for both populations. Over time, the relative proportion of low shELF3 vector CN clones dominates owing to the growth advantage provided by ELF3 expression. h Tumor growth curve of clonal populations of isogenic A549 shELF3 and control cells in NOD-SCID mice ( n = 24). i ELF3 expression of input clones as measured by immunoblot. j Control and absent shELF3 tumors at endpoint. k Cell viability as measured by annexin/PI staining of control (black bar) and clonal shELF3 (blue bar) LUAD cell lines cultured in complete (10% FBS, solid fill) or serum starved (0% FBS, hatched fill) media (H1993 n = 1 replicates owing to viability issues). l qPCR of ELF3 mRNA expression in control and shELF3 clonal cell lines with forced ELF3-OE or empty vector control (lacZ). Histograms summarizing the effect of ELF3 overexpression on colony formation, in terms of number m and size n of colonies formed (paired two-tailed Student’s t test). o Cell proliferation shows rescued growth rate with ELF3-OE in A549 cells (Wilcoxon test). p Tumor growth of shELF3.cl-lacZ and shELF3.cl-OE A549 cells in NRG mice ( n = 8). In all box and whisker plots, center line represents the median, box bounds indicate the 25th and 75th percentiles, and whiskers extend from minimum to maximum, all histograms display the mean + SEM ( n = 3 biological replicates unless otherwise stated). CN = copy number, + p < 0.10, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, paired two-tailed Student’s t test.

Article Snippet: Relative human cell-specific expression of ELF3 (Hs00963882_g1 – no cross reactivity with mouse) and NOTCH3 (Hs01128537_m1 – no cross reactivity with mouse) was quantified by qRT-PCR (TaqMan—Applied Biosystems) using mouse Actb (Mm04394036_g1) and human Actb (Hs99999903_m1) as endogenous controls.

Techniques: Expressing, shRNA, Inhibition, Over Expression, Knockdown, Control, Plasmid Preparation, Clone Assay, Western Blot, Staining, Cell Culture, Two Tailed Test, Whisker Assay

Journal: Nature Communications

Article Title: Epithelial tumor suppressor ELF3 is a lineage-specific amplified oncogene in lung adenocarcinoma

doi: 10.1038/s41467-019-13295-y

Figure Lengend Snippet: Pan-cancer analysis of ELF3 protein–protein interaction networks reveals tissue specificity. a Tissues are ordered vertically according to decreasing number of altered ELF3 PPIs. Disrupted PPIs were identified by comparing cancer profiles with non-malignant profiles in the same tissue types, with PPIs gained in cancer shown in red, and those lost in cancer shown in blue. The largest disrupted network was in lung cancer, with 111 PPIs gained and 85 PPIs lost in cancer. Considering all altered PPIs, 57% were specific to one tissue and only 15 PPIs were gained or lost in more than three tissues. b The largest disrupted network was in lung adenocarcinoma (LUAD), with 14 lost and 9 gained PPIs in cancer. The only altered PPI common to LUAD and squamous cell carcinoma (LUSC) was CCL11, whereas MYC, GLI2, and NKX2–1 were common to LUAD and large cell carcinoma (LULC). The protein names of the altered PPIs are indicated at the bottom of the figure. c Comparison of altered ELF3 PPIs between TCGA LUAD data and isogenic A549 cells (KRAS mutant samples only). Twenty-nine out of 156 PPI partners were significantly deregulated in LUAD with high ELF3 expression and are color-coded by their respective GO Molecular Function. Proteins at the left-side show significantly upregulated (up-triangles) mRNAs when ELF3 is highly expressed in TCGA, and right-side proteins represent significantly downregulated (down-triangles) mRNAs when ELF3 is highly expressed in TCGA. Edge color represents positive co-expression (red edges) or negative co-expression (blue edges), and edge thickness is proportional to the number of data sets supporting the edge. Node outline color represents up- or down-regulation in A549 control cells compared with clonal shELF3 cells ( n = 3 biological replicates). Therefore, bright green outline around up-triangles, and gray outline around down-triangles indicate consistency between TCGA and isogenic cell line data. Node size is proportional to the total number of altered PPIs, and node highlight size is proportional to the FDR corrected p value of expression fold change in isogenic cell lines, whereas blue bars indicate the fold change value. Circles in the center indicate ELF3 partners whose differential expression was not statistically significant. PPI = protein–protein interaction.

Article Snippet: Relative human cell-specific expression of ELF3 (Hs00963882_g1 – no cross reactivity with mouse) and NOTCH3 (Hs01128537_m1 – no cross reactivity with mouse) was quantified by qRT-PCR (TaqMan—Applied Biosystems) using mouse Actb (Mm04394036_g1) and human Actb (Hs99999903_m1) as endogenous controls.

Techniques: Comparison, Mutagenesis, Expressing, Control, Quantitative Proteomics

Journal: Nature Communications

Article Title: Epithelial tumor suppressor ELF3 is a lineage-specific amplified oncogene in lung adenocarcinoma

doi: 10.1038/s41467-019-13295-y

Figure Lengend Snippet: Prognostic relevance of ELF3 expression in non-small cell lung cancer. Kaplan–Meier survival curves comparing overall survival of lung cancer patients with high or low ELF3 expression (top and bottom tertiles, log-rank p values). All Stages: NSCLC n = 1926, p = 4.65E-04, Hazard Ratio (HR) (95% confidence interval) = 1.32 (1.13–1.55); LUAD n = 720, p = 5.09E-07, HR = 2.08 (1.57–2.76); LUSC n = 524, p = 0.229, HR = 1.20 (0.89–1.60). Stage I: NSCLC n = 577, p = 2.30E-05, HR = 2.11 (1.51–2.95); LUAD n = 370, p = 1.00E-06, HR = 3.66 (2.27–5.88); LUSC n = 172, p = 0.357, HR = 1.27 (0.76–2.14). NSCLC = non-small cell lung cancer; LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma.

Article Snippet: Relative human cell-specific expression of ELF3 (Hs00963882_g1 – no cross reactivity with mouse) and NOTCH3 (Hs01128537_m1 – no cross reactivity with mouse) was quantified by qRT-PCR (TaqMan—Applied Biosystems) using mouse Actb (Mm04394036_g1) and human Actb (Hs99999903_m1) as endogenous controls.

Techniques: Expressing