Journal: Cancer Discovery

Article Title: Convergent Genetic Adaptation in Human Tumors Developed Under Systemic Hypoxia and in Populations Living at High Altitudes

doi: 10.1158/2159-8290.CD-24-0943

Figure Lengend Snippet: Measurements of hypoxia-driven DNA damage and DNA damaging metabolites in PC12 PPGL tumor-derived cells, EPAS1 -mutated PPGL tumors and EPAS1 -overexpressing pheochromocytoma-derived cells ( EPAS1 -OE). A, Representative immunofluorescence images showing γH2AX foci formation and PARP1 expression in PC12 cells exposed to hypoxia (1% O 2 ) for 30 hours (short hypoxia) or 36 days (long hypoxia), or maintained under normoxic (21% O 2 ) conditions (left); quantification of the mean γH2AX foci per cell (right). B, Representative immunoblot images (above) and quantification of the indicated proteins (below) in PC12 cells exposed to normoxia or hypoxia for the specified timepoints. C, Comet assay analysis of DNA damage in PC12 cells exposed to normoxia (21% O 2 ) for 36 days or hypoxia (1% O 2 ) for 30 hours or 36 days (above); representative immunofluorescence images of comet tails:tail moment quantification. About 50–100 cells were scored per condition in each independent experiment (below). A higher percentage of DNA in the tail correlates with increased DNA damage. PC12 cells exposed to hypoxia for 30 hours exhibited significantly higher levels of DNA damage than cells cultured under normoxic conditions (15.75% vs. 11.80% tail DNA; P = 0.028). In addition, prolonged hypoxic exposure (up to 36 days) led to an even greater percentage of DNA in the comet tail (21.22% vs. 11.80% tail DNA; P = 0.0089), suggesting a deficiency in DNA repair mechanisms under these conditions. L, long; S, short. D, Elevated levels of fumarate metabolite in EPAS1 -mutated PPGL tumors and increased levels of succinate metabolite in SDH x-mutated PPGL tumors. Metabolite analyses of fumarate, succinate, and fumarate/succinate ratio in EPAS1 -mutated PPGL tumors ( n = 12, red) and VHL - and SDHx -mutated pseudohypoxic PPGL tumors ( EPAS1 -WT tumors, n = 129, blue) as already described . P values of the statistical significance between EPAS1 -mutated tumors vs EPAS1 -WT tumors were calculated using the Wilcoxon test. Supplementary Figure S12 shows the results of all P values calculated using the Wilcoxon test; only significant P values are indicated. E, Increased levels of fumarate/succinate metabolite in EPAS1 -OE cells. Metabolite analyses of fumarate, succinate, and fumarate/succinate ratio in MPCmCherry cells transfected with an overexpressing EPAS1 -HIF2α ( EPAS1 -OE, n = 8, red) and empty vector as control ( EPAS1 -null, empty vector, n = 8, blue) as described by Bechmann and colleagues . Supplementary Figure S15 shows the P values calculated using the Wilcoxon test. F, Model of DNA damage and oncometabolites implication in EPAS1 -mutated tumor PPGLs and PPGL cell models with EPAS1 mutation or cultured under long-term hypoxia conditions. OE, overexpression. ( F, Created with BioRender.com .)

Article Snippet: Briefly, cells were transfected using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) and HIF2α WT and HIF2α P405A/P531A plasmids (Addgene plasmid IDs #26055 and #19006, respectively).

Techniques: Derivative Assay, Immunofluorescence, Expressing, Western Blot, Single Cell Gel Electrophoresis, Cell Culture, Transfection, Plasmid Preparation, Control, Mutagenesis, Over Expression

Journal: Cancer Discovery

Article Title: Convergent Genetic Adaptation in Human Tumors Developed Under Systemic Hypoxia and in Populations Living at High Altitudes

doi: 10.1158/2159-8290.CD-24-0943

Figure Lengend Snippet: Transcription analyses for functional and developmental insights of EPAS1 in PPGL tumorigenesis. A, Workflow of bulk RNA-seq data analysis using three different cohorts: cohort I comprised non-CCHD sympathetic PPGL tumors: WT ( n = 46) or mutated ( n = 8) for EPAS1 ( , ); cohort II comprised sympathetic PPGL tumors from patients with hemoglobin disorders: 11 tumors EPAS1 WT and 19 tumors EPAS1 MUT ; cohort III comprised PPGL-derived cell line that we cultured under normoxic and hypoxic conditions for 12, 24, and 48 hours and 36 days (three replicates for each condition, 12 samples in normoxia and 12 in hypoxia). Of the 354 genes that were differentially expressed in patients with PPGL tumors, 14 mitochondrial genes were found, of which one was COX4I2 , the less common isoform of COX (mitochondrial respiratory complex 4) and the terminal electron acceptor of the oxidative phosphorylation system. B, Top part: Volcano plots of the differentially expressed genes between EPAS1 MUT and EPAS1 WT tumor samples from cohorts I and II, and between hypoxia and normoxia conditions in cohort III. Fourteen differentially expressed mitochondrial genes are highlighted (upregulated genes in orange color, downregulated genes in green color). Bottom part: normalized expression of COX4I2 in cohorts I, II, and III. C, Mitochondrial respiration using Seahorse equipment ( n = 6/group; mean ± SEM) in stable cell line HEK293T expressing empty vector (EV)–Control, HIF2α WT , and mutant HIF2α P405A/P531A through plasmid transfection. Oxygen consumption respiration (OCR; top), basal respiration (bottom). D, scRNA-seq data comprising the entire tree of neural crest lineage development, which includes differentiation trajectory from the neural crest toward chromaffin cells via Schwann cell precursor and bridge-state intermediates . The scRNA-seq data show that EPAS1 and COX4I2 expression are upregulated during development and downregulated in postnatal chromaffin/adrenal medulla cells, after the onset of breathing. BCCs, boundary cap cells; NCCs, neural crest cells; Th, tyrosine hydroxylase (chromaffin cell marker). ( A, Created with BioRender.com and D, Schematic of scRNA-seq was created with BioRender.com .)

Article Snippet: Briefly, cells were transfected using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific) and HIF2α WT and HIF2α P405A/P531A plasmids (Addgene plasmid IDs #26055 and #19006, respectively).

Techniques: Functional Assay, RNA Sequencing, Derivative Assay, Cell Culture, Phospho-proteomics, Expressing, Stable Transfection, Plasmid Preparation, Control, Mutagenesis, Transfection, Marker