Genome-wide identification and annotation of HIF-1α binding sites in two cell lines using massively parallel sequencing

We identified 531 and 616 putative HIF-1α target sites by ChIP-Seq in the cancerous cell line DLD-1 and the non-cancerous cell line TIG-3, respectively. We also examined the positions and expression levels of transcriptional start sites (TSSs) in these cell lines using our TSS-Seq method. We observed that 121 and 48 genes in DLD-1 and TIG-3 cells, respectively, had HIF-1α binding sites in proximal regions of the previously reported TSSs that were up-regulated at the transcriptional level. In addition, 193 and 123 of the HIF-1α target sites, respectively, were located in proximal regions of previously uncharacterized TSSs, namely, TSSs of putative alternative promoters of protein-coding genes or promoters of putative non-protein-coding transcripts. The hypoxic response of DLD-1 cells was more significant than that of TIG-3 cells with respect to both the number of target sites and the degree of induced changes in transcript expression. The Nucleosome-Seq and ChIP-Seq analyses of histone modifications revealed that the chromatin formed an open structure in regions surrounding the HIF-1α binding sites, but this event occurred prior to the actual binding of HIF-1α. Different cellular histories may be encoded by chromatin structures and determine the activation of specific genes in response to hypoxic shock. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11568-011-9150-9) contains supplementary material, which is available to authorized users

FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal

Puma is an essential mediator of p53-dependent and -independent apoptosis in vivo. In response to genotoxic stress, Puma is induced in a p53-dependent manner. However, the transcription factor driving Puma up-regulation in response to p53-independent apoptotic stimuli has yet to be identified. Here, we show that FOXO3a up-regulates Puma expression in response to cytokine or growth factor deprivation. Importantly, dysregulated Akt signaling in lymphoid cells attenuated Puma induction upon cytokine withdrawal. Our results suggest that Puma, together with another BH3 only member, Bim, function as FOXO3a downstream targets to mediate a stress response when PI3K/Akt signaling is down-regulated

Recommendations related to the analytical equivalence assessment of gene panel testing

Advances in cancer genome care over the past few years have included the development of gene panel testing for various biomarkers. This article summarizes issues and provides recommendations related to analytical performance evaluations for new oncology gene panels. The scope of these recommendations includes comprehensive genomic profiling assays related to gene panel testing that uses histological or serum specimens to detect gene mutations. As a research project of the Japan Agency for Medical Research and Development Research on Regulatory Science of Pharmaceuticals and Medical Devices, we convened the working group committee that consisted of more than 30 experts from academia, industry, and government. We have discussed the points that should be considered to allow maximal simplification of assessments using clinical specimens in evaluating accuracy and limit of detection in equivalence and analytical performance for three years. We provide recommendations specific to each type of gene mutation as well as to reference standards or specimens used for evaluations. In addition, in order to facilitate the discussion on the analytical performance of gene panel tests by multidisciplinary tumor boards of hospitals, the present recommendations also describe the items that companies are expected to provide information on in their packaging inserts and reports, and the items that are expected to be discussed by multidisciplinary tumor boards. Our working group document will be important for participants in multidisciplinary tumor boards including medical oncologists and genome scientists, and developers of gene panels not only in Japan but also in other countries

Metabolic Characterization of Antifolate Responsiveness and Non-responsiveness in Malignant Pleural Mesothelioma Cells

Antifolates are a class of drugs effective for treating malignant pleural mesothelioma (MPM). The majority of antifolates inhibit enzymes involved in purine and pyrimidine synthesis such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART). In order to select the most suitable patients for effective therapy with drugs targeting specific metabolic pathways, there is a need for better predictive metabolic biomarkers. Antifolates can alter global metabolic pathways in MPM cells, yet the metabolic profile of treated cells has not yet been clearly elucidated. Here we found that MPM cell lines could be categorized into two groups according to their sensitivity or resistance to pemetrexed treatment. We show that pemetrexed susceptibility could be reversed and DNA synthesis rescued in drug-treated cells by the exogenous addition of the nucleotide precursors hypoxanthine and thymidine (HT). We observed that the expression of pemetrexed-targeted enzymes in resistant MPM cells was quantitatively lower than that seen in pemetrexed-sensitive cells. Metabolomic analysis revealed that glycine and choline, which are involved in one-carbon metabolism, were altered after drug treatment in pemetrexed-sensitive but not resistant MPM cells. The addition of HT upregulated the concentration of inosine monophosphate (IMP) in pemetrexed-sensitive MPM cells, indicating that the nucleic acid biosynthesis pathway is important for predicting the efficacy of pemetrexed in MPM cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, and points to potential biomarkers for informing clinical decisions regarding the most effective therapies for patients with MPM

Association of variations in HLA class II and other loci with susceptibility to EGFR-mutated lung adenocarcinoma

Lung adenocarcinoma driven by somatic EGFR mutations is more prevalent in East Asians (30-50%) than in European/Americans (10-20%). Here we investigate genetic factors underlying the risk of this disease by conducting a genome-wide association study, followed by two validation studies, in 3,173 Japanese patients with EGFR mutation-positive lung adenocarcinoma and 15,158 controls. Four loci, 5p15.33 (TERT), 6p21.3 (BTNL2), 3q28 (TP63) and 17q24.2 (BPTF), previously shown to be strongly associated with overall lung adenocarcinoma risk in East Asians, were re-discovered as loci associated with a higher susceptibility to EGFR mutation-positive lung adenocarcinoma. In addition, two additional loci, HLA class II at 6p21.32 (rs2179920; P =5.1 × 10(-17), per-allele OR=1.36) and 6p21.1 (FOXP4) (rs2495239; P=3.9 × 10(-9), per-allele OR=1.19) were newly identified as loci associated with EGFR mutation-positive lung adenocarcinoma. This study indicates that multiple genetic factors underlie the risk of lung adenocarcinomas with EGFR mutations

AKT phosphorylation was induced under glucose deprivation.

<p>(A) Immunoblotting analyses after incubation of HepG2 cells in the absence or presence of 5.5 mM of glucose and absence or presence of 30 µM of LY294002 for the indicated times. (B) HepG2 cells treated or not treated with various concentrations of glucose for 0.5 h were subjected to immunoblotting. (C) Immunoblotting analyses of HepG2 cells treated or not treated with 5.5 mM of glucose, 5.5 mM of galactose, or 5.5 mM of fructose for 0.5 h. (D) Immunoblotting analyses of HepG2 cells treated or not treated with 5.5 mM of glucose, 5.5 mM of 2-DG, or 5.5 mM of glucose plus 5.5 mM of 2-DG for 0.5 h.</p

ROS mediates AKT phosphorylation under glucose deprivation.

<p>(A)(B)(D) HepG2 cells were cultured in either glucose-containing medium or glucose-deprived medium in the absence or presence of 12.5 mM of NAC for 0.5 h. ROS production was measured using flow cytometry. Cells were stained with (A) 5 µM of DCFDA or (B) 5 µM of BES-H₂O₂. Cells were gated within a range contained in the upper 5% of the total cell count under the glucose replete condition. (D) The AKT phosphorylation level was evaluated by immunoblotting. (C) Addition of H₂O₂ to media containing 5.5 mM of glucose in the absence or presence of 30 µM of LY294002 for 0.5 h, followed by immunoblotting.</p

SRC and OSSA are indispensable for the AKT phosphorylation induced by glucose deprivation.

<p>(A) Immunoblotting analyses of HepG2 cells in the absence or presence of 5.5 mM of glucose <u>in the</u> and absence or presence of 20 µM of PP2 for the indicated times. (B) Addition of H₂O₂ to the culture medium containing 5.5 mM glucosein the absence or presence of 20 µM of PP2 for 0.5 h, followed by immunoblotting. (C) HepG2 cells were cultured in medium containing or not containing (glucose-deprived) 5.5 mM of glucose in the absence or presence of 30 µM of LY204002 or 20 µM of PP2 for 0.5 h. The cells were stained with 5 µM of BES-H₂O₂. ROS production was measured using flow cytometry. (D) siRNA-treated HepG2 cells were subjected to immunoblotting analyses using OSSA antibody. (E) Immunoblotting analyses of HepG2 cells transfected with a non-targeting siRNA or two separate OSSA siRNAs in the absence or presence of 5.5 mM of glucose for the indicated times. (F) Addition of H₂O₂ to the medium of OSSA-knockdown cells containing 5.5 mM glucose for 0.5 h, followed by immunoblotting.</p