Transcriptomic and proteomic study of cancer cell lines exposed to actinomycin D and nutlin-3a reveals numerous, novel candidates for p53-regulated genes.

Transcriptomic analyses have revealed hundreds of p53-regulated genes; however, these studies used a limited number of cell lines and p53-activating agents. Therefore, we searched for candidate p53-target genes by employing stress factors and cell lines never before used in a high-throughput search for p53-regulated genes. We performed RNA-Seq on A549 cells exposed to camptothecin, actinomycin D, nutlin-3a, as well as a combination of actinomycin D and nutlin-3a (A + N). The latter two substances synergise upon the activation of selected p53-target genes. A similar analysis was performed on other cell lines (U-2 OS, NCI-H460, A375) exposed to A + N. To identify proteins in cell lysates or those secreted into a medium of A549 cells in control conditions or treated with A + N, we employed mass spectrometry. The expression of selected genes strongly upregulated by A + N or camptothecin was examined by RT-PCR in p53-deficient cells and their controls. We found that p53 participates in the upregulation of: ACP5, APOL3, CDH3, CIBAR2, CRABP2, CTHRC1, CTSH, FAM13C, FBXO2, FRMD8, FRZB, GAST, ICOSLG, KANK3, KCNK6, KLRG2, MAFB, MR1, NDRG4, PTAFR, RETSAT, TMEM52, TNFRSF14, TRANK1, TYSND1, WFDC2, WFDC5, WNT4 genes. Twelve of these proteins were detected in the secretome and/or proteome of treated cells. Our data generated new hypotheses concerning the functioning of p53. Many genes activated by A + N or camptothecin are also activated by interferons, indicating a noticeable overlap between transcriptional programs of p53 and these antiviral cytokines. Moreover, several identified genes code for antagonists of WNT/ß-catenin signalling pathways, which suggests new connections between these two cancer-related signalling systems. One of these antagonists is DRAXIN. Previously, we found that its gene is activated by p53. In this study, using mass spectrometry and Western blotting, we detected expression of DRAXIN in a medium of A549 cells exposed to A + N. Thus, this protein functions not only in the development of the nervous system, but it may also have a new cancer-related function.

The Wheel of p53 Helps to Drive the Immune System.

The p53 tumor suppressor protein is best known as an inhibitor of the cell cycle and an inducer of apoptosis. Unexpectedly, these functions of p53 are not required for its tumor suppressive activity in animal models. High-throughput transcriptomic investigations as well as individual studies have demonstrated that p53 stimulates expression of many genes involved in immunity. Probably to interfere with its immunostimulatory role, many viruses code for proteins that inactivate p53. Judging by the activities of immunity-related p53-regulated genes it can be concluded that p53 is involved in detection of danger signals, inflammasome formation and activation, antigen presentation, activation of natural killer cells and other effectors of immunity, stimulation of interferon production, direct inhibition of virus replication, secretion of extracellular signaling molecules, production of antibacterial proteins, negative feedback loops in immunity-related signaling pathways, and immunologic tolerance. Many of these p53 functions have barely been studied and require further, more detailed investigations. Some of them appear to be cell-type specific. The results of transcriptomic studies have generated many new hypotheses on the mechanisms utilized by p53 to impact on the immune system. In the future, these mechanisms may be harnessed to fight cancer and infectious diseases.

Effect of Escitalopram on the Number of DCX-Positive Cells and NMUR2 Receptor Expression in the Rat Hippocampus under the Condition of NPSR Receptor Blockade.

BACKGROUND: Neuropeptide S (NPS) is a multifunctional regulatory factor that exhibits a potent anxiolytic activity in animal models. However, there are no reports dealing with the potential molecular interactions between the activity of selective serotonin reuptake inhibitors (SSRIs) and NPS signaling, especially in the context of adult neurogenesis and the expression of noncanonical stress-related neuropeptides such as neuromedin U (NMU). The present work therefore focused on immunoexpression of neuromedin U receptor 2 (NMUR2) and doublecortin (DCX) in the rat hippocampus after acute treatment with escitalopram and in combination with selective neuropeptide S receptor (NPSR) blockade. METHODS: Studies were carried out on adult, male Sprague-Dawley rats that were divided into five groups: animals injected with saline (control) and experimental individuals treated with escitalopram (at single dose 10 mg/kg daily), escitalopram + SHA-68, a selective NPSR antagonist (at single dose 40 mg/kg), SHA-68 alone, and corresponding vehicle control. All animals were sacrificed under halothane anaesthesia. The whole hippocampi were quickly excised, fixed, and finally sliced for general qualitative immunohistochemical assessment of the NPSR and NMUR2 expression. The number of immature neurons was enumerated using immunofluorescent detection of doublecortin (DCX) expression within the subgranular zone (SGZ). RESULTS: Acute escitalopram administration affects the number of DCX and NMUR2-expressing cells in the adult rat hippocampus. A decreased number of DCX-expressing neuroblasts after treatment with escitalopram was augmented by SHA-68 coadministration. CONCLUSIONS: Early pharmacological effects of escitalopram may be at least partly connected with local NPSR-related alterations of neuroblast maturation in the rat hippocampus. Escitalopram may affect neuropeptide and DCX-expression starting even from the first dose. Adult neurogenesis may be regulated via paracrine neuropeptide S and NMU-related signaling.

Germline Variants in Angiogenesis-Related Genes Contribute to Clinical Outcome in Head and Neck Squamous Cell Carcinoma.

Fibroblast growth factor (FGF)/FGF receptor (FGFR), and platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) systems, as well as some matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), are involved in various steps of angiogenesis. Data indicate that common germline variations in angiogenesis-regulating genes may modulate therapy results and cancer progression. However, whether these variants affect clinical outcome in head and neck squamous cell carcinoma (HNSCC) is unclear. Hence, we assessed the relationship between FGF/FGFR, PDGF/PDGFR, MMP, and TIMP genetic variants and treatment outcomes in HNSCC patients receiving radiotherapy (RT) alone or combined with cisplatin-based chemotherapy. In multivariate analysis, FGF2 rs1048201 CC homozygotes showed a higher risk of death (p = 0.039), while PDGFRA rs2228230 T was strongly associated with an increased risk of locoregional relapse (HR 2.49, p = 0.001) in the combination treatment subgroup. In the RT alone subset, MMP2 rs243865 TT carriers had a higher risk of locoregional recurrence (HR 2.92, p = 0.019), whereas PDGFRB rs246395 CC homozygotes were at increased risk of metastasis (HR 3.06, p = 0.041). The MMP2 rs7201 C and TIMP2 rs7501477 T were associated with a risk of locoregional failure in the entire cohort (p = 0.032 and 0.045, respectively). Furthermore, rs1048201, rs2228230, rs246395, rs243865, rs7201, and rs7201/rs7501477 were independent indicators of an unfavorable outcome. This study demonstrates that the FGF2, PDGFRA, PDGFRB, MMP2, and TIMP2 variants may contribute to treatment failure and poor prognosis in HNSCC.

Activation of the atypical NF-κB pathway induced by ionizing radiation is not affected by the p53 status.

DNA double-strand breaks induced by ionizing radiation can activate the atypical NF-κB pathway via ATM-mediated phosphorylation of NEMO/IKKγ. We aimed to determine whether the status of p53 influenced the activation of this particular NF-κB pathway. The NF-κB signaling was activated either by irradiation with a single 8 Gy dose or by TNFα cytokine in p53-proficient and p53-deficient variants of HCT116, RKO, and U2-OS human cancer cell lines. To assess pathway activation the kinetics of phosphorylation (Ser32) and proteolytic degradation of IκBα inhibitor and phosphorylation (Ser536) of RelA(p65) NF-κB subunit were analyzed. Though activation of the radiation-induced atypical pathway was delayed and weakened when compared to the cytokine-induced canonical pathway, no significant differences were noted between p53-proficient and p53-deficient variants, which indicated that activation of both NF-κB pathways was not affected by the p53 status. In marked contrast, the presence of p53 significantly affected downstream effects of NF-κB activation, i.e. transcription of NF-κB-dependent genes. However, different patterns of such interference were observed, which indicated gene-specific and cell-specific mechanisms of interactions between NF-κB and p53 at the transcription regulation level.

Transcriptome Analysis of Cells Exposed to Actinomycin D and Nutlin-3a Reveals New Candidate p53-Target Genes and Indicates That CHIR-98014 Is an Important Inhibitor of p53 Activity.

Co-treatment with actinomycin D and nutlin-3a (A + N) strongly activates p53. Previously we reported that CHIR-98014 (GSK-3 kinase inhibitor), acting in cells exposed to A + N, prevents activation of TREM2-an innate immunity and p53-regulated gene associated with Alzheimer's disease. In order to find novel candidate p53-target genes and genes regulated by CHIR-98014, we performed RNA-Seq of control A549 cells and the cells exposed to A + N, A + N with CHIR-98014 or to CHIR-98014. We validated the data for selected genes using RT-PCR and/or Western blotting. Using CRISPR/Cas9 technology we generated p53-deficient cells. These tools enabled us to identify dozens of candidate p53-regulated genes. We confirmed that p53 participates in upregulation of BLNK, APOE and IRF1. BLNK assists in activation of immune cells, APOE codes for apolipoprotein associated with Alzheimer's disease and IRF1 is activated by interferon gamma and regulates expression of antiviral genes. CHIR-98014 prevented or inhibited the upregulation of a fraction of genes stimulated by A + N. Downregulation of GSK-3 did not mimic the activity of CHIR-98014. Our data generate the hypothesis, that an unidentified kinase inhibited by CHIR-98014, participates in modification of p53 and enables it to activate a subset of its target genes, e.g., the ones associated with innate immunity.

Synergistic activation of p53 by actinomycin D and nutlin-3a is associated with the upregulation of crucial regulators and effectors of innate immunity.

Actinomycin D and nutlin-3a (A + N) activate p53, partly through induction of phosphorylation on Ser392. The death of A549 cells induced by A + N morphologically resembles inflammation-inducing pyroptosis - cell destruction triggered by activated caspase-1. The treatment with A + N (or camptothecin) strongly upregulated caspase-1 and its two activators: IFI16 and NLRP1, however, caspase-1 activation was not detected. A549 cells may have been primed for pyroptosis, with the absence of a crucial trigger. The investigation of additional innate immunity elements revealed that A + N (or camptothecin) stimulated the expression of NLRX1, STING (stimulator of interferon genes) and two antiviral proteins, IFIT1 and IFIT3. IFI16 and caspase-1 are coded by p53-regulated genes which led us to investigate regulation of NLRP1, NLRX1, STING, IFIT1 and IFIT3 in p53-dependent mode. The upregulation of NLRP1, NLRX1 and STING was attenuated in p53 knockdown cells. The upsurge of the examined genes, and activation of p53, was inhibited by C16, an inhibitor of PKR kinase. PKR was tested due to its ability to phosphorylate p53 on Ser392. Surprisingly, C16 was active even in PKR knockdown cells. The ability of C16 to prevent activation of p53 and expression of innate immunity genes may be the source of its strong anti-inflammatory action. Moreover, cells exposed to A + N can influence neighboring cells in paracrine fashion, for instance, they shed ectodomain of COL17A1 protein and induce, in p53-dependent mode, the expression of gene for interleukin-7. Further, the activation of p53 also spurred the expression of SOCS1, an inhibitor of interferon triggered STAT1-dependent signaling. We conclude that, stimulation of p53 primes cells for the production of interferons (through upregulation of STING), and may activate negative-feedback within this signaling system by enhancing the production of SOCS1.