Journal: Molecular Cancer Research

Article Title: HIF Inactivation of p53 in Ovarian Cancer Can Be Reversed by Topotecan, Restoring Cisplatin and Paclitaxel Sensitivity

doi: 10.1158/1541-7786.mcr-18-1109

Figure Lengend Snippet: Figure 1. A, TPT inhibits HIF1a protein accumulation. OVCA429, CAOV3, TOV112D, and CAOV4 cells were incubated with or without TPT (25 and 100 nmol/L) for 48 hours followed by a 24-hour exposure to hypoxia (1% O2). B, TPT inhibits hypoxia-induced upregulation of VEGF protein secretion. Cell lines were pretreated with TPT for 48 hours and incubated under hypoxic conditions (1% O2) for 24 hours. Kinetic studies established 24 hours as optimal for VEGF upregulation (data not shown). Mean normoxic VEGF levels for CAOV3, TOV112D, and CAOV4 cells were 302, 414, and 502 pg/mL, respectively. Results are fraction of VEGF protein levels under normoxic conditions in the absence of TPT. Data represent average SD from three independent experiments. Analysis was performed using a one- tail unpaired t test; , P < 0.05; , P < 0.005. C, TPT upregulates p53 and BAX protein levels. OVCA429, CAOV3, CAOV4, and TOV112D cells were incubated with or without 25 and 100 nmol/L TPT with hypoxia for 48 hours, and p53 and BAX protein levels were evaluated via Western analysis.

Article Snippet: TOPO1-HIF1a mRNA in vitro–binding studies HIF1a mRNA was synthesized from human HIF1a cDNA (Origene) using the Ribomax Kit (Promega).

Techniques: Incubation, Western Blot

Journal: Molecular Cancer Research

Article Title: HIF Inactivation of p53 in Ovarian Cancer Can Be Reversed by Topotecan, Restoring Cisplatin and Paclitaxel Sensitivity

doi: 10.1158/1541-7786.mcr-18-1109

Figure Lengend Snippet: Figure 4. A and B, TPT-mediated reversal of hypoxia-induced drug resistance. CAOV3 cells were pretreated with sub-IC50 doses of TPT (25 nmol/L) for 4 hours followed by exposure to hypoxic conditions for 24 hours (0.5% O2). TPT- containing media were replaced with fresh media, and cells were treated for 72 hours with cisplatin (A) or paclitaxel (B) in triplicate in either hypoxic or normoxic conditions. Results are average SD from three independent experiments. TPT specifically inhibited HIF1a protein stabilization under hypoxic conditions.

Article Snippet: TOPO1-HIF1a mRNA in vitro–binding studies HIF1a mRNA was synthesized from human HIF1a cDNA (Origene) using the Ribomax Kit (Promega).

Techniques:

Journal: Molecular Cancer Research

Article Title: HIF Inactivation of p53 in Ovarian Cancer Can Be Reversed by Topotecan, Restoring Cisplatin and Paclitaxel Sensitivity

doi: 10.1158/1541-7786.mcr-18-1109

Figure Lengend Snippet: Figure 6. Effect of TOPO1 siRNA TPT on TOPO1 and HIF1a protein levels determined by Western blot and subcellular localization of TOPO1. A, CAOV3 cells were treated with Topo I siRNA (30 nmol/L) for 96 hours, followed by TPT (800 nmol/L) for 4 hours, followed by 4 hours of hypoxia (0.5% O2) and probed for TOPO1. Blots were also probed for HIF1a. Results shown are representative of three individual experiments performed. B, CAOV3 cells were pretreated with TOPO1 siRNA (30 nmol/L) for 48 hours and then exposed to various doses of cisplatin or paclitaxel for 72 hours hypoxia and tested for proliferation inhibition using XTT assay. Results are expressed as fraction of control.

Article Snippet: TOPO1-HIF1a mRNA in vitro–binding studies HIF1a mRNA was synthesized from human HIF1a cDNA (Origene) using the Ribomax Kit (Promega).

Techniques: Western Blot, Inhibition, XTT Assay, Control

Journal: Molecular Cancer Research

Article Title: HIF Inactivation of p53 in Ovarian Cancer Can Be Reversed by Topotecan, Restoring Cisplatin and Paclitaxel Sensitivity

doi: 10.1158/1541-7786.mcr-18-1109

Figure Lengend Snippet: Figure 7. TOPO1 binding to HIF1a mRNA determined using RIP and real-time PCR (A). For isolation of RNA–protein complexes from CAOV3 and OVCA429 cells, total RNA was extracted from mRNP lysate derived from CAOV3 cells immunoprecipitated with a monoclonal TOPO1 antibody and subjected to real-time PCR with two different primer sets specific for HIF1a (GenBank accession number, NM_001530.3): Primer-A: 50- GAAAG CGCAA GTCCT CAAAG-30 (sense) and 50- TGGGT AGGAG ATGGA GATGC-30 (antisense); Primer-B: 50-CCGCT GGAGA CACAA TCATA-30

Article Snippet: TOPO1-HIF1a mRNA in vitro–binding studies HIF1a mRNA was synthesized from human HIF1a cDNA (Origene) using the Ribomax Kit (Promega).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Isolation, Derivative Assay, Immunoprecipitation

Journal: Nature metabolism

Article Title: miR-147b-mediated TCA cycle dysfunction and pseudohypoxia initiate drug tolerance to EGFR inhibitors in lung adenocarcinoma

doi: 10.1038/s42255-019-0052-9

Figure Lengend Snippet: a , Fractional viability of H1975 3D structures treated with osimertinib (25 nM), LNA miR-147b inhibitor (LNA-anti147b, 90 nM), DMOG (10 μM) or combinations for 14 days. n=3 independent biological replicates. b , qRT-PCR analysis for hypoxia gene expression in H1975 cells treated with 90 nM LNA miR-147b inhibitor (LNA-anti147b) and 10 µM DMOG or vehicle for three days. The relative gene expression in scrambled control cells treated with vehicle was calibrated as 1. n=3 independent biological replicates. c , Fractional viability of H1975 3D structures treated with 25 nM osimertinib, 90 nM LNA-anti147b, 30 µM R59949 or combinations for 14 days. n=7 independent biological replicates. d, qRT-PCR analysis of HIF1A in H1975 cells with shRNAs against HIF1A . H1975 cells were transfected with shRNAs against HIF1A (shHIF1A-1 and -2) or scrambled control (shCtrl) and selected with 0.5 μg/ml puromycin. GAPDH was used as endogenous control. n=3 independent biological replicates. e , Cell viability of H1975 cells with HIF1A knockdown treated with osimertinib. The cells with shRNAs against HIF1A (shHIF1A-1 and shHIF1A-2) and scrambled control cells (shCtrl) were treated with 100 nM osimertinib or vehicle for 3 days. The cell viability was analyzed on day 4. n=4 independent biological replicates. f , Cell viability of H1975 cells with constitutive active HIF1A mutant treated with osimertinib. The cells were transfected with HIF1A A588T and scrambled control cells (Scr) followed by 600 μg/ml neomycin selection. Then the cells were treated with 100 nM osimertinib or vehicle for 3 days. The cell viability was analyzed on day 4. n=4 independent biological replicates. g, Derivation and growth of 3D structures from lung PDX tumors. (top) Representative phase contrast microscopy for parental EGFR mutant lung PDX-derived 3D structures in PDX_LU_10 3D structures. Repeated six times with similar results. (Bottom) growth curve of PDX 3D structures. The 3D structures size was measured every two days. The media were replenished every three days till day 14. n=3 independent biological replicates. Scale bar, 50 µm. h , Pretreatment response on lung PDX_LU_10 3D structures with LNA miR-147b inhibitor (anti147b) and osimertinib. The 3D structures were established at medium size seven days after seeding 2000 single-cells into 3D cultures in 96-well plate. This timepoint was recorded as day 0. Then the 3D structures were administrated with LNA anti147b or antictrl (90 nM) on day 0 and day 2 or osimertinib (25 nM) on day 1 and day 4. The vehicle treated group did not receive treatments with LNA or osimertinib. The 3D structures’ size was measured every two days. The media were replenished every three days till day 14. n=3 independent biological replicates. i , Schematic for miR-147b-driven drug-tolerance model. miR-147b is enriched in a subpopulation of parental lung cancer cells entering drug-tolerant status when they are treated with EGFR-TKIs. miR-147b mediates drug-tolerance through repressing activities of VHL and SDH leading to activated pseudohypoxia response. TKI, tyrosine kinase inhibitor; SDH, succinate dehydrogenase; TCA, tricarboxylic acid; PHD, prolyl-hydroxylase. Data are mean ± s.e.m and were analysed with Kruskal-Wallis test ( a,c ); unpaired two-tailed t -test ( b,d,e,f,h).

Article Snippet: A mixture of 2.5 μg pGFP-C-shLenti vector targeting HIF1A (OriGene, Cat #320380), EPAS1 (OriGene, Cat #TL315484), scrambled negative control (Cat #TR30021), lentiviral vector targeting HIF1A mutant A588T (OriGene, Cat #RC402571), control vector and 7.5 µL of PureFection were used for transfection.

Techniques: Quantitative RT-PCR, Expressing, Transfection, Mutagenesis, Selection, Microscopy, Derivative Assay, Two Tailed Test