Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet: REIMS Analysis Predicts Breast Cancer Molecular Markers Including Oncogenic Mutations in PIK3CA (A) Schematic overview of sample preparation for REIMS analysis. (B) Area under the curve (AUC) classification accuracies for ER, PR, HER2 receptor, and triple negative status of 43 breast cancer (BC) cell lines (median intensity of n = 3 biological replicates) following feature selection for phospholipids in the m/z range 600–900, and leave-one-out cross validation. (C) Immunoblot analysis of estrogen inducible protein pS2 and predicted ESR1 expression in ER +ve MCF7 cells following treatment with 0.1% DMSO or indicated concentrations of 4-OHT for 72 h. (D) Unsupervised hierarchical clustering of 872 lipid species detected by REIMS across 43 BC cell lines. (E) Dendrogram of BC cell lines and isogenic MCF10A cells harboring either WT or MUT (E545K or H1047R) PIK3CA. (F) Immunoblot analysis of mature SREBP1 transcription factor expression in nuclear extracts of the MCF10A PIK3CA isogenic panel. (G) Relative exogenous fatty acid uptake in MCF10A PIK3CA WT and MUT cells following serum starvation for 1 h and supplementation with fluorescently labeled dodecanoic acid (n = 5 replicates). (H and I) Unsupervised hierarchical clustering of 9 PIK3CA WT and 9 MUT breast PDX tumors (H) and (I) 5 WT and 7 MUT primary breast tumors. Individual rows in the heatmaps in (D), (H) and (I) correspond to scaled Z score phospholipid intensities (n = 3 biological replicates). Error bars represent ± SEM. n.s., not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001. p values in (C, bottom panel) and (G) were calculated with one-way ANOVA, followed by unpaired, two-tailed Student’s t test with Bonferroni correction.

Article Snippet: SKBR3 (human breast carcinoma) , ATCC , Cat# HTB-30; RRID: CVCL_0033.

Techniques: Sample Prep, Selection, Biomarker Discovery, Western Blot, Expressing, Labeling, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet: Dietary Supplementation of Arachidonic Acid Reverses the Sensitivity of PIK3CA Mutant Tumors to cPLA2 Inhibition (A) Schematic of in vivo experimental design and profiling of breast cancer cell line xenografts with REIMS. (B and C) Relative tumor growth of CAL-51 ( PIK3CA MUT)-derived xenografts stably expressing control shGFP or two independent shRNAs targeting cPLA2 (cPLA2-sh1 and cPLA2-sh5) under (B) fat-free or (C) “Western” diets. (D) Weights of tumors excised at the end of the experiments (B) and (C). (E and F) Relative tumor growth of Hs578T ( PIK3CA WT)-derived xenografts stably expressing control shGFP or two independent shRNAs targeting cPLA2 under (E) fat-free or (F) “Western” diets. (G) Weights of tumors excised at the end of the experiments (E) and (F). (H) Representative images of H&E staining from resected tumors in (D) and (G). The black masks in (H) represent viable tumor area, while unshaded regions correspond to necrotic tissue. (I and J) Quantification of viable tumor area from (I) PIK3CA MUT (CAL-51) and (J) PIK3CA WT (Hs578T) tumor sections based on the analysis depicted in (H). (K and L) AA levels measured by REIMS in (K) PIK3CA MUT (CAL-51) and (L) PIK3CA WT (Hs578T) snap frozen excised tumors. AA intensities are reported as scaled values to the appropriate shGFP-fat-free diet condition. Data in (B), (C), (E), (F), (K), and (L) represent the mean ± SEM of relative tumor growth or tumor REIMS measurements from n = 3–5 mice. n.s., not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; p values in (B), (C), (E), and (F) were calculated using two-way ANOVA, and one-way ANOVA followed by unpaired, two-tailed Student’s t test with Bonferroni correction was used in (D), (G), and (I)–(L).

Article Snippet: SKBR3 (human breast carcinoma) , ATCC , Cat# HTB-30; RRID: CVCL_0033.

Techniques: Mutagenesis, Inhibition, In Vivo, Derivative Assay, Stable Transfection, Expressing, Control, Western Blot, Staining, Two Tailed Test

Journal: Cell

Article Title: Metabolic Fingerprinting Links Oncogenic PIK3CA with Enhanced Arachidonic Acid-Derived Eicosanoids

doi: 10.1016/j.cell.2020.05.053

Figure Lengend Snippet:

Article Snippet: SKBR3 (human breast carcinoma) , ATCC , Cat# HTB-30; RRID: CVCL_0033.

Techniques: Produced, Virus, Recombinant, Transfection, Protease Inhibitor, Lysis, Mutagenesis, Proliferation Assay, In Situ, Calcium Assay, Reverse Transcription, Kinase Assay, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Proximity Ligation Assay, CRISPR, Control, shRNA, Amplification, Plasmid Preparation, Positive Control, Software, Modification, Targeted Proteomics, Mass Spectrometry, Western Blot