Journal: NPJ Breast Cancer

Article Title: FOXA1 and adaptive response determinants to HER2 targeted therapy in TBCRC 036

doi: 10.1038/s41523-021-00258-0

Figure Lengend Snippet: a Schematic overview of LCCC1214 (Clinical trials identifier NCT01875666) for the analysis of adaptive responses to HER2 targeted therapy. b Normalized RNAseq data from matched patient samples was used to generate log2 fold changes for expressed genes (post-treatment vs. pre-treatment). The fold change in expression was used for the principal component analysis (PCA). Labels indicate patient number and dot color indicates treatment arm. The blue dashed line indicates distinct matched samples (from patients 116, 119, and 123) determined to be strongly responsive based on their distinct expression changes. c Log 2 fold change for FOXA1 for matched patient samples is plotted. The blue dashed line indicates the strongly responsive sample pairs. d Log 2 fold changes of the top 5000 differentially expressed genes for matched patient samples were used for unsupervised hierarchical clustering and nearest neighbor analysis was performed for the FOXA1 . The top 10 genes by Pearson correlation are indicated, as well as select genes identified from HER2+ cell line SE analysis. e The breast invasive carcinoma (TCGA, provisional) data set was used to identify genes most significantly co-expressed with HER3 expression in patient tumors. The top genes were XBP1 (not shown) and FOXA1 . The dashed line indicates regression line. Table contains legend for mutation status, if known. f Pathway analysis of gene expression data from matched patient samples was performed and the pathway scores differences was used for marker selection comparing the strongly responsive sample pairs (blue dashed line) to all other sample pairs. The top 25 pathways (increased and decreased) are shown (10% FDR cutoff). Representative significant pathways are listed with associated reference.

Article Snippet: Equal amounts of extracted protein (determined by Bradford assay) were separated by SDS-PAGE, transferred to nitrocellulose and probed with anti-BRD4 (Bethyl Laboratories, Cat. A301-985A100, 1:1000), anti-ERK2 (Santa Cruz Cat. sc-1647, 1:2000), anti-FOXA1 (Abcam, Cat. ab5089, 1:1000) or the following antibodies (all with Cell Signaling Cat. numbers and used at 1:1000): anti-FOXO1 (2880), anti-FOXO3 (2497), anti-phospho-HER2/ErbB2 (Tyr1221/1222) (2243), anti-HER2 (2242), anti-HER3 (4754), anti-phospho-AKT (Thr308) (4056), anti-phospho-AKT (Ser473) (4060), anti-phospho-ERK1/2 (4370).

Techniques: Expressing, Mutagenesis, Marker, Selection

Journal: NPJ Breast Cancer

Article Title: FOXA1 and adaptive response determinants to HER2 targeted therapy in TBCRC 036

doi: 10.1038/s41523-021-00258-0

Figure Lengend Snippet: a All post-treatment samples with RNAseq data were used to filter for expressed kinases. The normalized kinome expression data was used for principal component analysis (PCA). Patient number is indicated in red for each dot and color indicates LCCC1214 treatment arm. Dashed line indicates previously identified patients 116, 119, and 123 and neighboring samples 109, 118, and 121. b RNAseq expression levels of strongly responsive post-treatment samples were compared to the less-responsive post-treatment samples by DESeq2 to identify differentially expressed kinases. The volcano plot indicates the magnitude and the significance of the identified expression differences. Dashed line indicates FDR 5%. c Log 2 normalized gene expression data (GSE76360) of the kinome was analyzed from the 03-311 clinical trial (Clinical trials identifier NCT00148668), in which patient samples were obtained pre-treatment and after 10–14 days of trastuzumab. Post-treatment gene expression data of the kinome was used for unsupervised hierarchical clustering and a segregated cluster of 7 post-treatment patient samples (of 50 total) was identified. Marker selection was performed in Morpheus (Broad Institute) to identify the top 20 kinases significantly down in the strongly responsive post-treatment samples for 03-311 trial data and from DESeq2 analysis of the LCCC1214 trial from ( b ). Venn diagram shows the 6 kinases common to both data sets. Asterisks indicate that more than one gene probe was identified by marker selection for the 03-311 data set (Illumina expression beadchip). d DESeq2 analysis of differentially expressed transcription factors in the strongly responsive versus weakly responsive post-treatment LCCC1214 samples. The volcano plot indicates the magnitude and the significance of the identified expression differences. e Marker selection was performed in Morpheus (Broad Institute) to identify the top 20 transcription factors with decreased expression in the strongly responsive post-treatment samples for 03-311 trial and compared with the DESeq2 analysis for LCCC1214 from ( a ). The Venn diagram shows the 5 transcription factors common to both clinical data sets. f The post-treatment/pre-treatment log 2 fold change of HER2 , HER3 , and PTK6 expression is shown for paired patient samples. g Paired post-treatment and pre-treatment samples from the LCCC1214 study were processed for kinome analysis by MIB/MS. The log 2 MIB binding changes (post/pre) were plotted as the sum of strongly molecular responsive samples (patients 109, 116, 119, and 121) versus the average difference for weakly-responsive matched pairs and the top 25 differences in both directions are shown. h – j Log 2 fold changes for the indicated matched patient samples as determined by MIB binding ( x -axis) and RNAseq ( y -axis).

Article Snippet: Equal amounts of extracted protein (determined by Bradford assay) were separated by SDS-PAGE, transferred to nitrocellulose and probed with anti-BRD4 (Bethyl Laboratories, Cat. A301-985A100, 1:1000), anti-ERK2 (Santa Cruz Cat. sc-1647, 1:2000), anti-FOXA1 (Abcam, Cat. ab5089, 1:1000) or the following antibodies (all with Cell Signaling Cat. numbers and used at 1:1000): anti-FOXO1 (2880), anti-FOXO3 (2497), anti-phospho-HER2/ErbB2 (Tyr1221/1222) (2243), anti-HER2 (2242), anti-HER3 (4754), anti-phospho-AKT (Thr308) (4056), anti-phospho-AKT (Ser473) (4060), anti-phospho-ERK1/2 (4370).

Techniques: Expressing, Marker, Selection, Binding Assay