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.

BRCA1/BRCA2 variants of uncertain significance in clinical practice: A case report.

The influence of BRCA1/2 variants of uncertain significance (VUSs) on the cancer risk and their association with the response to treatment is uncertain. The aim of the present study was to evaluate the role of BRCA VUS in patients with breast cancer. A total of two cases of breast cancer patients with the BRCA VUS were described. The complete coding sequence of BRCA1/2 genes was analyzed from the genomic DNA material by next generation sequencing on the Ion Torrent platform. The presence of c.3454G>A (p.Asp1152Asn) VUS in the BRCA1 gene was reported in a 64-year-old woman with invasive breast carcinoma. The characteristics of the breast tumors were the following: moderately differentiated-intermediate grade (NG-2 G-2), HER2 (+), estrogen receptor (ER) (+++), progesterone receptor (PR) (+++), luminal A subtype and pT2 N1a Mx. The second detected VUS was the c.2374T>C (p.Tyr792His) variant in the BRCA2 gene. This variant was reported in a 33-year-old woman who was diagnosed with right breast cancer (cT2N1M0). The invasive breast carcinoma was characterized as follows: NG-2 G-2, ER (+++), PR (+++), Ki-67 10%, HER2 (+++) and luminal B subtype. The data demonstrated that patients with VUSs should be managed based on their family history of cancer and clinicopathological characteristics. The clinical significance of the VUS in BRCA1/2 may change over time and reclassification of the variant to 'pathogenic' or 'benign' should be undertaken. Patients with VUS should be followed up regularly.

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.

PIM2 survival kinase is upregulated in a p53-dependent manner in cells treated with camptothecin or co-treated with actinomycin D and nutlin-3a.

The p53 protein is an inducer of apoptosis, acting as a transcriptional regulator of apoptotic genes. In a previous study, we found that actinomycin D and nutlin-3a (A + N) synergistically activate p53. To better understand the molecular consequences of this synergism, we incubated arrays of antibodies against apoptotic proteins with extracts of A549 cells in which p53 had been activated. We found that strong activation of p53, marked by serine 46 and 392 phosphorylation, was associated with inactivating phosphorylation of proapoptotic BAD protein on serine 136. Investigation of the source of this phosphorylation revealed that activation of p53 was associated with accumulation of PIM2, a survival kinase. The accumulation of PIM2 following treatment with A + N was suppressed in p53-knockdown cells. Others discovered that PIM2 was activated by cooperatively acting p53 molecules. Our results are consistent with this finding. Moreover, we found that in A549 cells, the treatment with A + N stimulated in p53-dependent fashion the expression of other high cooperativity p53 target genes, DRAXIN and H19. Activation of antiapoptotic H19 can mechanistically explain relatively low rate of apoptosis of A549 cells exposed to A + N. We conclude that PIM2, DRAXIN and H19 are efficiently stimulated by strongly activated p53 molecules, probably acting cooperatively.

The Alzheimer's disease-associated TREM2 gene is regulated by p53 tumor suppressor protein.

TREM2 mutations evoke neurodegenerative disorders, and recently genetic variants of this gene were correlated to increased risk of Alzheimer's disease. The signaling cascade originating from the TREM2 membrane receptor includes its binding partner TYROBP, BLNK adapter protein, and SYK kinase, which can be activated by p53. Moreover, in silico identification of a putative p53 response element (RE) at the TREM2 promoter led us to hypothesize that TREM2 and other pathway elements may be regulated in p53-dependent manner. To stimulate p53 in synergistic fashion, we exposed A549 lung cancer cells to actinomycin D and nutlin-3a (A + N). In these cells, exposure to A + N triggered expression of TREM2, TYROBP, SYK and BLNK in p53-dependent manner. TREM2 was also activated by A + N in U-2 OS osteosarcoma and A375 melanoma cell lines. Interestingly, nutlin-3a, a specific activator of p53, acting alone stimulated TREM2 in U-2 OS cells. Using in vitro mutagenesis, chromatin immunoprecipitation, and luciferase reporter assays, we confirmed the presence of the p53 RE in TREM2 promoter. Furthermore, activation of TREM2 and TYROBP by p53 was strongly inhibited by CHIR-98014, a potent and specific inhibitor of glycogen synthase kinase-3 (GSK-3). We conclude that TREM2 is a direct p53-target gene, and that activation of TREM2 by A + N or nutlin-3a may be critically dependent on GSK-3 function.

Actinomycin D and nutlin-3a synergistically promote phosphorylation of p53 on serine 46 in cancer cell lines of different origin.

The p53 tumor suppressor protein is a transcription factor activated by phosphorylation of its N-terminus. MDM2, encoded by a p53-activated gene, acts as a negative-feedback regulator of p53 by promoting p53 degradation. Moreover, MDM2 inhibits p53 by binding to and concealing its N-terminal transcription-activating domain. p53 can be activated by nutlin-3a, a molecule designed to bind MDM2 and prevent its interaction with p53. Actinomycin D promotes phosphorylation and accumulation of p53 via a mechanism that involves high expression of MDM2. We hypothesized that co-treatment of cells with actinomycin D and nutlin-3a would lead to synergistic activation of p53 by stimulating kinases and preventing accumulated MDM2 from binding to p53. Indeed, co-treatment of various cell lines with actinomycin D and nutlin-3a resulted in a synergistic increase of p53 phosphorylation on serine 46. We focused on this residue because it is a marker of the highest level of p53 activation. Co-treatment was associated with conspicuous decrease in a marker of mTOR activity in NCI-H28 cells and very strong activation of p53 targets, including CDKN1A and PML, in A549 cells. Other p53 target genes (SESN1, SESN2, TIGAR, DRAM1) were also efficiently upregulated; however, a marker of apoptosis (active caspase-3) appeared only in some cancer cell lines (e.g., A375 and other cell lines derived from melanoma) indicating that phosphorylation of p53 on serine 46 is not straightforwardly associated with induction of apoptosis. Moreover, our data suggest that melanoma may be a suitable target for drug combination used in this study.

Rapamycin prevents strong phosphorylation of p53 on serine 46 and attenuates activation of the p53 pathway in A549 lung cancer cells exposed to actinomycin D.

The activation of the p53 pathway by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a molecule that mimics metabolic stress, is attenuated by rapamycin, an inhibitor of mTOR kinase, immunosuppressant, and cancer drug. Rapamycin also extends lifespan in experimental animals. Because AICAR is a relatively weak activator of p53, we investigated whether stimulation of p53 by the strong activator actinomycin D is also sensitive to the inhibitory effect of rapamycin. In A549 lung cancer cells, activation of p53 by actinomycin D was associated with phosphorylation of p53 on Ser46. Rapamycin inhibited the accumulation of phospho-Ser46 p53, attenuated upregulation of some p53 target genes, and altered cell-cycle progression. Moreover, in cells exposed to actinomycin D, rapamycin attenuated the accumulation of PML, a protein that in some conditions stimulates Ser46 phosphorylation. However, Ser46 phosphorylation was not diminished in PML-knockdown cells, suggesting that in our system PML does not play a major role in stimulating p53 phosphorylation on Ser46. Knockdown of p53 diminished the upregulation of PML by stress-inducing agents, consistent with the idea that PML is a p53-regulated gene. Our data suggest that the attenuation of p53 phosphorylation on Ser46 may play a significant role in the biological activity of anti-aging rapamycin.

Nutlin-3a, an MDM2 antagonist and p53 activator, helps to preserve the replicative potential of cancer cells treated with a genotoxic dose of resveratrol.

Resveratrol is a natural compound that has been intensely studied due to its role in cancer prevention and potential as an anti-cancer therapy. Its effects include induction of apoptosis and senescence-like growth inhibition. Here, we report that two cancer cell lines (U-2 OS and A549) differ significantly in their molecular responses to resveratrol. Specifically, in U-2 OS cells, the activation of the p53 pathway is attenuated when compared to the activation in A549 cells. This attenuation is accompanied by a point mutation (458: CGA→TGA) in the PPM1D gene and overexpression of the encoded protein, which is a negative regulator of p53. Experimentally induced knockdown of PPM1D in U-2 OS cells resulted in slightly increased activation of the p53 pathway, most clearly visible as stronger phosphorylation of p53 Ser37. When treated with nutlin-3a, a non-genotoxic activator of p53, U-2 OS and A549 cells both responded with substantial activation of the p53 pathway. Nutlin-3a improved the clonogenic survival of both cell lines treated with resveratrol. This improvement was associated with lower activation of DNA-damage signaling (phosphorylation of ATM, CHK2, and histone H2AX) and higher accumulation of cells in the G1 phase of the cell cycle. Thus, the hyperactivation of p53 by nutlin-3a helps to preserve the replicative potential of cells exposed to resveratrol.

The activation of the p53 pathway by the AMP mimetic AICAR is reduced by inhibitors of the ATM or mTOR kinases.

A balanced diet reduces the risk of life-threatening diseases such as diabetes and cancer. A reduced supply of energy at the cellular level leads to an increased concentration of AMP, which, in turn, results in LKB1-mediated activation of the AMPK kinase. The activation of the p53 tumor suppressor protein by metabolic stress has been shown to be mediated by AMPK. Increased intracellular AMP can be mimicked by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). We showed that AICAR activated the p53 pathway in LKB1-deficient cells. This activation was strongly attenuated by two inhibitors of the ATM kinase (caffeine and Ku-55933), which is dysfunctional in ataxia-telanagiectasia patients. In cells with ATM expression silenced by shRNA, AICAR-induced p53 phosphorylation at Ser(15) and Ser(37) was attenuated. Furthermore, p53 activation by AICAR was blocked by rapamycin, a specific inhibitor of the mTOR kinase, which is a crucial regulator of cell growth. Rapamycin did not block p53 activation by resveratrol, which, in contrast to AICAR, induced the DNA damage response, senescence-like growth inhibition, a high level of post-translational modification of p53, and weak upregulation of MDM2 (the negative regulator of p53). Thus, ATM and mTOR participate in the activation of p53 in response to a compound mimicking metabolic stress.

Resveratrol induces senescence-like growth inhibition of U-2 OS cells associated with the instability of telomeric DNA and upregulation of BRCA1.

Resveratrol decreases cancer risk and improves health of laboratory animals. However, it can also promote genomic instability. Part of the beneficial activity of resveratrol may result from the activation of SIRT1 deacetylase. We examined how resveratrol influenced the growth of human cancer cell lines of different origin: osteosarcoma (U-2 OS) and lung adenocarcinoma (A549) and how it modulated the expression as well as the localization of key proteins, involved in DNA repair and cell cycle regulation. Resveratrol-induced growth arrest was associated with signs of stress-induced senescence. Differential expression of BRCA1, cyclin B1, pRb and p21 in U-2 OS and A549 cells indicates that resveratrol can engage various molecular mechanisms to arrest cell cycle progression. In subset of U-2 OS cells, the upregulated BRCA1 formed foci closely associated with WRN and the telomeric protein (TRF1). Moreover, resveratrol induced telomeric instability in U-2 OS cells and the activation of DNA damage signaling in both cell lines, manifested as the phosphorylation of histone H2AX at serine 139 and of p53 at serines 15 and 37. Our data are consistent with the hypothesis that resveratrol inhibits cell growth and induces senescence by altering DNA metabolism.