Journal: Molecular cancer research : MCR

Article Title: MAP3K7 loss drives enhanced androgen signaling and independently confers risk of recurrence in prostate cancer with joint loss of CHD1

doi: 10.1158/1541-7786.MCR-20-0913

Figure Lengend Snippet: (A) Western blot of shControl, shMAP3K7, shCHD1, and shMAP3K7-shCHD1 (shDouble) LNCaP cells. Multiple shRNA constructs are shown. (B) 5-day growth assay for LNCaP shControl, shMAP3K7, shCHD1, and various shDouble combinations in steroid-depleted medium with vehicle (0.1% EtOH) or 10 pM R1881. Asterick denotes shDouble (2) combination used in subsequent experiments. (n=3; p-value indicated for all shDouble constructs versus shControl at Day 5). (C) Quantification of day 5 from growth assay in B, including the shDouble-2 combination (n=3; p-values indicate comparison to shControl EtOH). (D) Day 7 of R1881 dose response curves for LNCaP cells normalized to respective EtOH treatment (n=3; p-values indicate comparison to shControl at each dose). (E) RT-qPCR of indicated genes in LNCaP cells cultured in steroid-depleted medium ± EtOH or 1 nM R1881 for 8 hr. Ct values normalized to B2M then to shControl (n=3; p-values represent comparison to shControl EtOH). (F) Quantification (n=3) and representative western blot of AR protein in shControl and shDouble LNCaP cell lysates isolated after different treatments: complete growth medium or steroid-depleted medium ± 1 nM R1881 for 4 hr or 24 hr. (G) Quantification (n=3) and representative western blot of AR protein in nuclear fractions isolated from LNCaP cell lysates after treatment with ± 1 nM R1881 for 4 hr. AR blot of EtOH-treated samples is shown at a higher exposure than R1881-treated samples in order to visualize bands. All data represent mean ± SEM; One-way ANOVA with Tukey’s multiple comparisons test (C, G), two-way ANOVA with Dunnett’s multiple comparisons test (B, D, E), and unpaired two-tailed t-test (F); *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: LAPC4 cells were also supplemented with 1 nM R1881 (Perkin Elmer, #NLP005005MG).

Techniques: Western Blot, shRNA, Construct, Growth Assay, Comparison, Quantitative RT-PCR, Cell Culture, Isolation, Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: MAP3K7 loss drives enhanced androgen signaling and independently confers risk of recurrence in prostate cancer with joint loss of CHD1

doi: 10.1158/1541-7786.MCR-20-0913

Figure Lengend Snippet: (A) Day 5 of growth assay for indicated LNCaP cells treated with 5-60 µM enzalutamide in complete medium, normalized to DMSO vehicle control (n=3; p-values indicate comparison to shControl at each concentration). (B) RT-qPCR of AR-FL and AR-v7 in indicated LNCaP cells ± 1 nM R1881 for 4 h. (C) RT-qPCR analysis of AR-v7 target genes UGT2B17, AKT1, and UBE2C in shControl and shDouble LNCaP cells ± EtOH or 1 nM R1881 for 4 h. For (B-C), Ct values normalized to B2M expression and then to shControl (n=3; p-values indicate comparison to shControl EtOH). (D) Correlation of UGT2B17, (E) AKT1, and (F) UBE2C mRNA expression with quartiles of MAP3K7 and CHD1 mRNA co-expression in the MSKCC dataset (25). (G) Propidium iodide (PI) counts normalized to cell confluence of shControl (left) and shDouble (right) LNCaP cells in castration conditions beginning 24 hours after transfection with indicated siRNAs (n=3; p-values indicate comparison to siCtrl). (H) Western blot of lysates from LNCaP cells 96 hours after transfection with indicated siRNAs showing cleaved PARP, total PARP, and the loading control β-actin. All data represent means ± SEM; unpaired two-tailed t-tests (D, E, F) and two-way ANOVA with Dunnett’s multiple comparisons test (A, B, C, G); *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: LAPC4 cells were also supplemented with 1 nM R1881 (Perkin Elmer, #NLP005005MG).

Techniques: Growth Assay, Control, Comparison, Concentration Assay, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: MAP3K7 loss drives enhanced androgen signaling and independently confers risk of recurrence in prostate cancer with joint loss of CHD1

doi: 10.1158/1541-7786.MCR-20-0913

Figure Lengend Snippet: (A) Schematic of experimental setup and workflow for RNA-Seq and ChIP-seq. (B) Heatmap illustration of RNA-Seq z-score mRNA expression changes of a validated AR target gene signature in indicated cell types ± 1 nM R1881 for 4 hr (33). (C) GSEA of AR signaling gene signature (MSigDB: M15861) in indicated TCGA PCa groups with copy number loss of MAP3K7, CHD1, or both versus PCa with loss of neither. (D) Normalized signal intensity (read count per million mapped reads) of AR ChIP-seq binding events centered on the peak regions in indicated cells treated with vehicle EtOH (top) or 1 nM R1881 (bottom). (E) Venn diagram of differential, gene-annotated AR ChIP binding sites in indicated cell types treated with 1 nM R1881 compared to shControl EtOH as a background (fold-change >4, p-value <1e-04). (F) Venn diagram of the annotated AR ChIP sites from (E) that are further filtered for significant gene expression changes (FDR<0.05) over shControl EtOH. (G) Scatter plots of annotated genes from (F) including the shControl R1881 versus shControl EtOH comparison not shown in (F). Genes with log2 fold-change ± 1.5 are shown in green.

Article Snippet: LAPC4 cells were also supplemented with 1 nM R1881 (Perkin Elmer, #NLP005005MG).

Techniques: RNA Sequencing, ChIP-sequencing, Expressing, Binding Assay, Gene Expression, Comparison

Journal: Molecular cancer research : MCR

Article Title: MAP3K7 loss drives enhanced androgen signaling and independently confers risk of recurrence in prostate cancer with joint loss of CHD1

doi: 10.1158/1541-7786.MCR-20-0913

Figure Lengend Snippet: (A) Pathway enrichment analysis for LNCaP shMAP3K7 versus shControl (+ R1881) RNA-Seq changes using Metascape (28). (B) Summary of GSEA results comparing TCGA cases with lower quartile mRNA expression of MAP3K7, CHD1 or both versus cases with upper quartile expression of respective groups. Nominal p-value <0.05 for all enrichments included. (C) GSEA plots of signatures particularly enriched in TCGA cases with low versus upper quartile expression of MAP3K7. (D) GSEA plots of the G2M signature in TCGA MAP3K7 low versus high cases and LNCaP shMAP3K7 versus shControl cells (+R1881). (E) Flow cytometry cell cycle analysis of indicated LNCaP cells after 48 hours in steroid-depleted medium ± 1 nM R1881 showing percent of total cells in G2M (n=3). Data represent mean ± SEM. Log-rank Mantel-Cox test (A, B), One-way ANOVA with Tukey’s multiple comparisons test (G); *=p<0.05; **=p<0.01; ***=p<0.001; ****=p<0.0001.

Article Snippet: LAPC4 cells were also supplemented with 1 nM R1881 (Perkin Elmer, #NLP005005MG).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Cell Cycle Assay

Journal: Therapeutic Advances in Medical Oncology

Article Title: Combination of tissue and liquid biopsy molecular profiling to detect transformation to small cell lung carcinoma during osimertinib treatment

doi: 10.1177/1758835920974192

Figure Lengend Snippet: Alteration landscape of the paired tumor tissue biopsies collected before (yellow headings) and after histological transformation (green headings). The numbers in the boxes correspond to the variant allele frequency of the mutation or the gene copy number for amplifications. Samples were analyzed using the Advanta Solid Tumor NGS Library Prep Assay (Fluidigm) and sequenced on a NextSeq platform (Illumina). LC, lung carcinoma.

Article Snippet: Libraries were prepared using the Advanta Solid Tumor NGS Library Prep Assay with the automated Juno TM system on integrated fluidic circuits (LP 8.8.6 IFC) (Fluidigm) following the manufacturer’s procedure.

Techniques: Transformation Assay, Variant Assay, Mutagenesis