Oxygen tension-dependent variability in the cancer cell kinome and response to combination therapies
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE244662 | GEO | 2024/06/03
REPOSITORIES: GEO
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Project description:Solid tumors are characterized by significantly lower oxygen (O2) levels. Despite this fact, routine preclinical cancer studies are performed under ambient O2 conditions. We have developed an experimental approach that allows us to collect, process and evaluate cancer and non-cancer cells under physioxia (3-5% O2), in such a way that there is very minimal exposure to air. We have shown in previous studies that ambient and physioxic O2 differentially impact relevant cell surface markers and sensitivity to therapy. In this study, our goal was to further explore oxygen-dependent signaling pathway alterations and to determine how these pathways influence response to targeted therapies. Using cells derived from transgenic mouse mammary tumors and healthy primary human breast epithelial cells, we show the impact of ambient air and physioxia on the kinome, with subsequent pathway alterations that mediate the effectiveness of single and combination therapies. Our data emphasize the importance of O2 considerations in preclinical cancer research and drug development.
2024-05-08 | PXD046005 | Pride
Project description:Preclinical studies of primary cancer cells are always done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3-10% range, with most tumors in an hypoxic or 1-2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anti-cancer agents is routinely examined at ambient O2. Here, using both mouse models and human cancers, we demonstrate that tumors collected, processed and propagated at physiologic (physioxia) O2 compared to ambient air display very distinct differences in key signaling networks including Lgr5/Wnt, Yap, and Nrf2/Keap1, nuclear reactive oxygen species levels, alternative splicing, and sensitivity to several targeted therapies including PIK3CAalpha-specific and EGFR inhibitors. Significance: Extra-physiologic oxygen shock/stress (EPHOSS), as noted in cells collected/processed under ambient air, has been demonstrated to have significant impact on numbers and engrafting ability of hematopoietic stem cells. We report deleterious effects of EPHOSS on cancer cell behavior and EPHOSS-mediated effects on cancer cells give misleading information on signaling pathway activation that could severely impact the relevance of these findings. Cancer cells under EPHOSS show higher proliferation rate compared to cells under physioxia and thus are sensitive to anti-proliferative agents. Thus, drugs that show effectiveness on cancer cells collected in ambient air and subjected to EPHOSS may not be effective or as relevant in vivo, results that could partially explain the limited clinical translation of laboratory findings. Evaluating cell signaling and effects of drugs on cancer cells under physiologic O2 prior to in vivo studies could substantially reduce cost and aid in drug discovery relevant to the actual physioxia/pathological status of the tumor cells in vivo.
2022-02-16 | PXD023133 | Pride
Project description:The central nervous system normally functions at O2 levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O2-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O2 tensions compared to the cell culture standard of 20% O2, to investigate their ability to sense and translate this O2 information to transcriptional activity. Variance of ambient O2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O2 tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional ‘programs’ may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity.
2012-09-20 | GSE29296 | GEO
Project description:The central nervous system normally functions at O2 levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O2-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O2 tensions compared to the cell culture standard of 20% O2, to investigate their ability to sense and translate this O2 information to transcriptional activity. Variance of ambient O2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O2 tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional ‘programs’ may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity. Cerebral cortices were removed from 6-8-day-old Wistar rats, cortical astrocytes were isolated and these cells were maintained in a humidified incubator at 37°C (95% air; 5% CO2). This resulted in a culture of cortical astrocytes, as confirmed by visual inspection the following day and later by glial fibrillary acidic protein immunohistochemistry. Any cortical astrocyte culture that was not homogenous was disposed of and not used in this study. Culture medium was exchanged every 7 days and cells were grown in culture for up to 14 days. 24hr prior to experimentation cells were transferred to an hypoxic workstation equilibrated with either 1%, 4%, 9% or 21% O2 and 5% CO2, with the remaining percentage gas being N2. Once cortical astrocytes had reached approximately 90% confluence (75cm2 flask) they were subjected to hypoxia as above, washed with PBS, removed from the flask base with 0.05% trypsin-EDTA (Gibco) and then gently centrifuged (500xg). The cell pellet was re-suspended in PBS, centrifuged twice more to remove any traces of media, then triturated in 8-10 volumes of RNAlater (Applied Biosystems), frozen and stored at -80oC. Messenger RNA was amplified and labeled with biotin using the standard Illumina protocol (Illumina TotalPrep RNA Amplification Kit Ambion; Austin, TX, cat # IL1791), and hybridized to Illumina's Sentrix Rat Ref-12,v1 Expression BeadChips (Illumina, San Diego, CA). Three replicates from each treatment group (1%, 21%, 4%, and 9% O2) were hybridized and the data was extracted using the Illumina BeadStudio software(v3.4).
2012-09-20 | E-GEOD-29296 | biostudies-arrayexpress
Project description:Hematopoietic stem cells (HSCs) manifest impaired recovery and self-renewal with a concomitant increase in progenitor content (HPCs) when exposed to ambient air as opposed to physioxia. Mechanisms behind this distinction are poorly understood but have the potential to enhance HSC function for bone marrow transplantation. High resolution transcriptomic analysis revealed that HSCs under physioxia (HSCs- P) upregulate genes involved in self-renewal and quiescence but downregulate genes involved in apoptosis and differentiation compared to HSCs-A. Remarkably, among several genes, Tet2 was significantly downregulated in HSC-Ps compared to HSC-As, which was associated with reduced 5-hydroxymethylcytosine (5-hmC) levels in HSC-Ps. Interestingly, individuals carrying the TET2 mutation in their HSCs are at a higher risk of developing clonal hematopoiesis, myeloid leukemias and cardiovascular disease. Loss of Tet2 in mice results in more HSCs and enhanced self-renewal. TET enzymes are α-ketoglutarate, iron and oxygen-dependent dioxygenases that convert 5-methylcytosine to 5-hydroxymethylcytosine thereby promoting active transcription. We evaluated whether physioxia affects the numbers and function of HSCs and HPCs from Tet2-/- mice similar to wildtype (WT) mice. In contrast to WT-HSCs, we observed complete non-responsiveness of Tet2-/- HSCs to changes in oxygen tension. Tet2-/- HSCs and HPCs exhibited similar numbers and function under physioxia and ambient air. The lack of functional responsiveness to changes in oxygen tension in Tet2-/- HSCs was associated with similar changes in genes involved in self-renewal and quiescence among WT-HSC-Ps, Tet2-/- HSC-Ps and Tet2-/- HSC-As. Our findings define a novel molecular program involving Tet2 in regulating the self-renewal of HSCs under physioxia.
2022-01-07 | GSE192992 | GEO
Project description:Hematopoietic stem cells (HSCs) manifest impaired recovery and self-renewal with a concomitant increase in progenitor content (HPCs) when exposed to ambient air as opposed to physioxia. Mechanisms behind this distinction are poorly understood but have the potential to enhance HSC function for bone marrow transplantation. High resolution transcriptomic analysis revealed that HSCs under physioxia (HSCs- P) upregulate genes involved in self-renewal and quiescence but downregulate genes involved in apoptosis and differentiation compared to HSCs-A. Remarkably, among several genes, Tet2 was significantly downregulated in HSC-Ps compared to HSC-As, which was associated with reduced 5-hydroxymethylcytosine (5-hmC) levels in HSC-Ps. Interestingly, individuals carrying the TET2 mutation in their HSCs are at a higher risk of developing clonal hematopoiesis, myeloid leukemias and cardiovascular disease. Loss of Tet2 in mice results in more HSCs and enhanced self-renewal. TET enzymes are α-ketoglutarate, iron and oxygen-dependent dioxygenases that convert 5-methylcytosine to 5-hydroxymethylcytosine thereby promoting active transcription. We evaluated whether physioxia affects the numbers and function of HSCs and HPCs from Tet2-/- mice similar to wildtype (WT) mice. In contrast to WT-HSCs, we observed complete non-responsiveness of Tet2-/- HSCs to changes in oxygen tension. Tet2-/- HSCs and HPCs exhibited similar numbers and function under physioxia and ambient air. The lack of functional responsiveness to changes in oxygen tension in Tet2-/- HSCs was associated with similar changes in genes involved in self-renewal and quiescence among WT-HSC-Ps, Tet2-/- HSC-Ps and Tet2-/- HSC-As. Our findings define a novel molecular program involving Tet2 in regulating the self-renewal of HSCs under physioxia.
2022-01-07 | GSE192988 | GEO
Project description:Plants were grown in liquid media at ambient air composition [0.03% CO2, 78.97% N2, 21% O2] for 12 days. On 13th day one set (perturbed set) of samples (20 samples) were exposed to air with elevated CO2 composition [1% CO2, 78% N2, 21% O2] and the other set (control set) of samples (20 samples) were continued to grow at air of ambient composition. Two samples (shake flasks containing plants) were harvested at 1, 3, 6, 9, 12, 18, 24 and 30 hours from each of the two sets after the start of the treatment. From each of the control and perturbed set 4 samples were harvested to serve as pre-treatment controls.
2006-08-31 | E-MEXP-773 | biostudies-arrayexpress
Project description:The effect of CD151 expression onto the kinome of Jurkat T cells was assessed using kinome analysis. CD151 was expressed in Jurkat T cells by retroviral transduction based on a pMSCV vector. Entrez Gene: 977 UniProtKB: P48509 Jurkat T cells were transduced with the MSCV-CD151 vector and successfully transduced cells were selected using puromycin. For the kinome array experiments 3 independent samples of Jurkat cells and three independent samples of J-CD151 cells were collected. To minimize unspecific background signals, lysates from Jurkat and J-CD151 T cells harvested at different growth stages, which were then pooled to provide one sample prior to loading on the Kinexus antibody microarrays.
2017-09-30 | E-MTAB-5978 | biostudies-arrayexpress
Project description:The human kinome is incolved in multiple function in the life cycle of cells, and ther differntial expression in cacner suggests that protein kinases play an important role in tumor progression and proliferation. To delineate pathways that may be important for neoplastic change in women at high risk for ovarian cancer, we compared the expression signature of surface kinases in normal ovarian surface epithelium with ovarian epithelium from patients at high risk, and epithelial ovarian cancer using Affymetrix expresion array HG U133Plus2.
2009-04-07 | GSE15578 | GEO
Project description:Small molecule BET bromodomain inhibitors (BETi) are actively being pursued in clinical trials for the treatment of a variety of cancers, however, the mechanisms of resistance to targeted BET protein inhibitors remain poorly understood. Using a novel mass spectrometry approach that globally measures kinase signaling at the proteomic level, we evaluated the response of the kinome to targeted BET inhibitor treatment in a panel of BRD4-dependent ovarian carcinoma (OC) cell lines. Despite initial inhibitory effects of BETi, OC cells acquired resistance following sustained treatment with the BETi, JQ1. Through application of Multiplexed Inhibitor Beads (MIBs) and mass spectrometry, we demonstrate that BETi resistance is mediated by adaptive kinome reprogramming, where activation of compensatory pro-survival kinase networks overcomes BET protein inhibition. Furthermore, drug combinations blocking these kinases may prevent or delay the development of drug resistance and enhance the efficacy of BET inhibitor therapy. RNAseq was employed to identify changes in kinase RNA expression following short term (48h) or chronic (JQ1R) JQ1 treatment in three different ovarian cancer cell lines.
2016-07-21 | E-GEOD-82329 | biostudies-arrayexpress