P53 Activation In Response Research Articles

Targeting BRIX1 via Engineered Exosomes Induces Nucleolar Stress to Suppress Cancer Progression.

Elevated ribosome biogenesis correlates with the rapid growth and progression of cancer. Targeted blockade of ribosome biogenesis induces nucleolar stress, which preferentially leads to the elimination of malignant cells. In this study, it is reported that the nucleolar protein BRIX1 is a critical regulator for the homeostasis between ribosome biogenesis and p53 activation. BRIX1 facilitated the processing of pre-rRNA by supporting the formation of the PeBoW complex. In addition, BRIX1 prevented p53 activation in response to nucleolar stress by impairing the interactions between MDM2 and the ribosomal proteins, RPL5, and RPL11, thereby triggering the resistance of cancer cells to chemotherapy. Conversely, depletion of BRIX1 induced nucleolar stress, which in turn activated p53 through RPL5 and RPL11, consequently inhibiting the growth of tumors. Moreover, engineered exosomes are developed, which are surface-decorated with iRGD, a tumor-homing peptide, and loaded with siRNAs specific to BRIX1, for the treatment of cancer. iRGD-Exo-siBRIX1 significantly suppressed the growth of colorectal cancer and enhanced the efficacy of 5-FU chemotherapy in vivo. Overall, the study uncovers that BRIX1 functions as an oncoprotein to promote rRNA synthesis and dampen p53 activity, and also implies that targeted inhibition of BRIX1 via engineered exosomes can be a potent approach for cancer therapy.

Diamond-Blackfan anemia, the archetype of ribosomopathy: How distinct is it from the other constitutional ribosomopathies?

Diamond-Blackfan anemia (DBA) was the first ribosomopathy described in humans. DBA is a congenital hypoplastic anemia, characterized by macrocytic aregenerative anemia, manifesting by differentiation blockage between the BFU-e/CFU-e developmental erythroid progenitor stages. In 50 % of the DBA cases, various malformations are noted. Strikingly, for a hematological disease with a relative erythroid tropism, DBA is due to ribosomal haploinsufficiency in 24 different ribosomal protein (RP) genes. A few other genes have been described in DBA-like disorders, but they do not fit into the classical DBA phenotype (Sankaran et al., 2012; van Dooijeweert et al., 2022; Toki et al., 2018; Kim et al., 2017 [1–4]).Haploinsufficiency in a RP gene leads to defective ribosomal RNA (rRNA) maturation, which is a hallmark of DBA. However, the mechanistic understandings of the erythroid tropism defect in DBA are still to be fully defined. Erythroid defect in DBA has been recently been linked in a non-exclusive manner to a number of mechanisms that include: 1) a defect in translation, in particular for the GATA1 erythroid gene; 2) a deficit of HSP70, the GATA1 chaperone, and 3) free heme toxicity. In addition, p53 activation in response to ribosomal stress is involved in DBA pathophysiology. The DBA phenotype may thus result from the combined contributions of various actors, which may explain the heterogenous phenotypes observed in DBA patients, even within the same family.

Regulation of p53 by the mitotic surveillance/stopwatch pathway: implications in neurodevelopment and cancer.

The transcription factor p53 (encoded by TP53) plays diverse roles in human development and disease. While best known for its role in tumor suppression, p53 signaling also influences mammalian development by triggering cell fate decisions in response to a wide variety of stresses. After over 4 decades of study, a new pathway that triggers p53 activation in response to mitotic delays was recently identified. Termed the mitotic surveillance or mitotic stopwatch pathway, the USP28 and 53BP1 proteins activate p53 in response to delayed mitotic progression to control cell fate and promote genomic stability. In this Minireview, I discuss its identification, potential roles in neurodevelopmental disorders and cancer, as well as explore outstanding questions about its function, regulation and potential use as a biomarker for anti-mitotic therapies.

Association of p53 with Neurodegeneration in Parkinson's Disease.

p53 is a vital transcriptional protein implicated in regulating diverse cellular processes, including cell cycle arrest, DNA repair, mitochondrial metabolism, redox homeostasis, autophagy, senescence, and apoptosis. Recent studies have revealed that p53 levels and activity are substantially increased in affected neurons in cellular and animal models of Parkinson's disease (PD) as well as in the brains of PD patients. p53 activation in response to neurodegenerative stress is closely associated with the degeneration of dopaminergic neurons accompanied by mitochondrial dysfunction, reactive oxygen species (ROS) production, abnormal protein aggregation, and impairment of autophagy, and these pathogenic events have been implicated in the pathogenesis of PD. Pathogenic p53 integrates diverse cellular stresses and activate these downstream events to induce the degeneration of dopaminergic neurons; thus, it plays a crucial role in the pathogenesis of PD and appears to be a potential target for the treatment of the disease. We reviewed the current knowledge concerning p53-dependent neurodegeneration to better understand the underlying mechanisms and provide possible strategies for PD treatment by targeting p53.

A noninvasive iRFP713 p53 reporter reveals dynamic p53 activity in response to irradiation and liver regeneration in vivo.

Genetically encoded probes are widely used to visualize cellular processes in vitro and in vivo. Although effective in cultured cells, fluorescent protein tags and reporters are sub-optimal in vivo due to poor tissue penetration and high background signal. Luciferase reporters offer improved signal-to-noise ratios but require injections of luciferin that can lead to variable responses and that limit the number and timing of data points that can be gathered. Such issues in studying the critical transcription factor p53 have limited insight on its activity in vivo during development and tissue injury responses. Here, by linking the expression of the near-infrared fluorescent protein iRFP713 to a synthetic p53-responsive promoter, we generated a knock-in reporter mouse that enabled non-invasive, longitudinal analysis of p53 activity in vivo in response to various stimuli. In the developing embryo, this model revealed the timing and localization of p53 activation. In adult mice, the model monitored p53 activation in response to irradiation and paracetamol-or CCl4- induced liver regeneration. After irradiation, we observed potent and sustained activation of p53 in the liver, which limited the production of reactive oxygen species (ROS) and promoted DNA damage resolution. We propose that this new reporter may be used to further advance our understanding of various physiological and pathophysiological p53 responses.

Selected Articles from This Issue

Glioblastomas (GBM) are highly invasive brain tumors that can spread rapidly throughout the brain. GBM are marked by a high rate of disease recurrence after therap. by virtue of the self-renewal capacity of glioma stem cells (GSCs), which are highly heterogeneous. Here, Thomas and colleagues identify a subset of GSCs bearing copy number alterations of the ASNS gene, leading to elevated expression of asparagine synthase. ASNS-high GSCs bore distinct metabolic features from other GSCs, including increased capacity for both glycolysis and oxidative phosphorylation and the ability to switch between them as needed in response to environmental cues. This metabolic plasticity allowed for rapid dissemination of ASNS-high GSCs throughout the brain tissue and a marked resistance to oxidative stress, rendering these cells de novo resistant to radiation. These findings nominate ASNS as a potential therapeutic target for GBM, which could restrict the pathogenicity of GSCs and enhance the efficacy of standard treatment modalities.Inhibitors of poly (ADP-ribose) polymerase (PARPi) are known to be effective in treating a range of tumor types, particularly as a synthetic-lethal approach for tumors with defects in the homologous recombination DNA repair pathway. However, dose-limiting toxicities, particularly hematological toxicities, can cause early cessation of treatment and loss of therapeutic benefit. Here, using a CRISPR/Cas9 screen, Xu and colleagues identify the CHK2-p53 pathway as a key mediator of hematological toxicity in response to the clinically approved PARPi agent, olaparib. CHK2 was required for efficient activation of p53 in response to PARPi in blood cells, and genetic approaches that blocked this connection allowed cells to better tolerate olaparib treatment. Concurrent inhibition of CHK2 alongside olaparib emulated this protective effect, but importantly did not affect PARPi-mediated killing of ovarian cancer cell lines. If this finding extends to people, then these data nominate CHK2 inhibition as a potential target to spare patients from hematological adverse effects in response to PARPi, which could broaden the window of therapeutic benefit for these agents.Analogous to epigenetic control of gene expression through histone methylation, epitranscriptomic modifications such as N6-methyladenosine (m6A) function to modulate RNA stability, processing, and translation. In this study, Cotter and colleagues profile the activity of METTL3, a key constituent of the methyltransferase complex that catalyzes m6A modifications, and its role in establishing the pathologic transcriptome observed in metastatic castration-resistant prostate cancer (mCRPC). The authors observe that ablation of METTL3—and by extension, m6A modifications—is associated with resistance to androgen receptor signaling inhibitors, a key therapeutic modality in the clinical management of prostate cancer. This effect was shown to be mediated independently of the androgen signaling pathway, and instead relied on activation of a gastrointestinal-specific transcriptional signature driven by the expression of the nuclear receptor NR5A2/LRH-1. Taken together, these observations elucidate a novel mechanism by which epitranscriptomic m6A modifications may play a role in establishing the mCRp. transcriptome and in promoting disease progression.Pancreatic ductal adenocarcinoma (PDAC) patients often present with metastatic disease and face a dismal prognosis. Because of the high rate of metastasis observed in PDAC tumors, systemic therapies are frequently the only viable option for clinical management; however, PDAC lacks an effective armamentarium of targeted interventions, and tumors quickly develop resistance to standard chemotherapeutic regimens. Using single-cell sequencing of mouse PDAC tumors, Du and colleagues identify the receptor tyrosine kinase AXL as a mediator of PDAC progression and metastatic spread. The authors demonstrate that AXL is overexpressed in PDAC tumor cells with a mesenchymal phenotype, and that its activity promotes and is required for epithelial-mesenchymal transition and metastasis in vivo. Concordantly, PDAC tumors that developed in AXL-deficient mice were less aggressive, better differentiated, and more sensitive to gemcitabine chemotherapy than PDAC in mice bearing wild-type AXL. These data suggest that targeting AXL expression and/or activity may present an opportunity to suppress or treat PDAC progression.

DNA damage and oxidant stress activate p53 through differential upstream signaling pathways

Stabilization and activation of the p53 tumor suppressor are triggered in response to various cellular stresses, including DNA damaging agents and elevated Reactive Oxygen Species (ROS) like H2O2. When cells are exposed to exogenously added H2O2, ATR/CHK1 and ATM/CHK2 dependent DNA damage signaling is switched on, suggesting that H2O2 induces both single and double strand breaks. These collective observations have resulted in the widely accepted model that oxidizing conditions lead to DNA damage that subsequently mediates a p53-dependent response like cell cycle arrest and apoptosis. However, H2O2 also induces signaling through stress-activated kinases (SAPK, e.g., JNK and p38 MAPK) that can activate p53. Here we dissect to what extent these pathways contribute to functional activation of p53 in response to oxidizing conditions. Collectively, our data suggest that p53 can be activated both by SAPK signaling and the DDR independently of each other, and which of these pathways is activated depends on the type of oxidant used. This implies that it could in principle be possible to modulate oxidative signaling to stimulate p53 without inducing collateral DNA damage, thereby limiting mutation accumulation in both healthy and tumor tissues.

EGFR overexpression increases radiotherapy response in HPV-positive head and neck cancer through inhibition of DNA damage repair and HPV E6 downregulation.

High-risk Human Papillomavirus (HPV) infections have recently emerged as an independent risk factor in head and neck squamous cell carcinoma (HNSCC). There has been a marked increase in the incidence of HPV-induced HNSCC subtype, which demonstrates different genetics with better treatment outcome. Despite the favourable prognosis of HPV-HNSCC, the treatment modality, consisting of high dose radiotherapy (RT) in combination with chemotherapy (CT), remains similar to HPV-negative tumours, associated with toxic side effects. Epidermal growth factor receptor (EGFR) is overexpressed in over 80% of HNSCC and correlates with RT resistance. EGFR inhibitor Cetuximab is the only FDA approved targeted therapy for both HNSCC subtypes, however the response varies between HNSCC subtypes. In HPV-negative HNSCC, Cetuximab sensitises HNSCC to RT improving survival rates. To reduce adverse cytotoxicity of CT, Cetuximab has been approved for treatment de-escalation of HPV-positive HNSCC. The results of several recent clinical trials have concluded differing outcome to HPV-negative HNSCC. Here we investigated the role of EGFR in HPV-positive HNSCC response to RT. Remarkably, in HPV-positive HNSCC cell lines and in vivo tumour models, EGFR activation was strongly indicative of increased RT response. In response to RT, EGFR activation induced impairment of DNA damage repair and increased RT response. Furthermore, EGFR was found to downregulate HPV oncoproteinE6 expression and induced p53 activity in response to RT. Collectively, our data uncovers a novel role for EGFR in virally induced HNSCC and highlights the importance of using EGFR-targeted therapies in the context of the genetic makeup of cancer.

FATS regulates polyamine biosynthesis by promoting ODC degradation in an ER\u03b2-dependent manner in non-small-cell lung cancer

Polyamine biosynthesis is an essential metabolic pathway for cell growth and differentiation in non-small-cell lung cancer (NSCLC). Fragile-site associated tumour suppressor (FATS) is a novel gene involved in cancer. The results of our previous study showed that FATS-mediated polyubiquitination of p53 promotes the activation of p53 in response to DNA damage; however, little is known about the role of FATS in metabolic reprogramming in NSCLC. In the present study, FATS was observed to be significantly downregulated in NSCLC tissues compared with paired adjacent normal tissues and was associated with the survival of NSCLC patients. We further showed that the presence of the tumour suppressor FATS in NSCLC cells led to apoptosis by inducing pro-death autophagy. In addition, FATS was shown to function as a suppressor of polyamine biosynthesis by inhibiting ornithine decarboxylase (ODC) at the protein and mRNA levels, which was partially dependent on oestrogen receptor (ER). Furthermore, FATS was observed to bind to ERβ and translocate to the cytosol, leading to ODC degradation. The findings of our study demonstrate that FATS plays important roles in polyamine metabolism in NSCLC and provides a new perspective for NSCLC progression.

Sanggenol L Induces Apoptosis and Cell Cycle Arrest via Activation of p53 and Suppression of PI3K/Akt/mTOR Signaling in Human Prostate Cancer Cells.

Prostate cancer is the most common cancer in Western countries. Recently, Asian countries are being affected by Western habits, which have had an important role in the rapid increase in cancer incidence. Sanggenol L (San L) is a natural flavonoid present in the root barks of Morus alba, which induces anti-cancer activities in ovarian cancer cells. However, the molecular and cellular mechanisms of the effects of sanggenol L on human prostate cancer cells have not been elucidated. In this study, we investigated whether sanggenol L exerts anti-cancer activity in human prostate cancer cells via apoptosis and cell cycle arrest. Sanggenol L induced caspase-dependent apoptosis (up-regulation of PARP and Bax or down-regulation of procaspase-3, -8, -9, Bid, and Bcl-2), induction of caspase-independent apoptosis (up-regulation of AIF and Endo G on cytosol), suppression of cell cycle (down-regulation of CDK1/2, CDK4, CDK6, cyclin D1, cyclin E, cyclin A, and cyclin B1 or up-regulation of p53 and p21), and inhibition of PI3K/Akt/mTOR signaling (down-regulation of PI3K, p-Akt, and p-mTOR) in prostate cancer cells. These results suggest the induction of apoptosis via suppression of PI3K/Akt/mTOR signaling and cell cycle arrest via activation of p53 in response to sanggenol L in prostate cancer cells.

SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway.

MDM2 is an oncogene and critical negative regulator of tumor suppressor p53. Genotoxic stress causes alternative splicing of MDM2 transcripts, which leads to alterations in p53 activity and contributes to tumorigenesis. MDM2-ALT1 is one of the alternatively spliced transcripts predominantly produced in response to genotoxic stress, and is comprised of terminal coding exons 3 and 12. Previously, we found that SRSF1 induces MDM2-ALT1 by promoting MDM2 exon 11 skipping. Here we report that splicing regulator SRSF2 antagonizes the regulation of SRSF1 by facilitating the inclusion of exon 11 through binding at two conserved exonic splicing enhancers. Overexpression of SRSF2 reduced the generation of MDM2-ALT1 under genotoxic stress, whereas SRSF2 knockdown induced the expression of MDM2-ALT1 in the absence of genotoxic stress. Blocking the exon 11 SRSF2-binding sites using oligonucleotides promoted MDM2-ALT1 splicing and induced p53 protein expression, and apoptosis in p53 wild-type cells. The regulation of MDM2 splicing by SRSF2 is also conserved in mice, as mutation of one SRSF2-binding site in Mdm2 exon 11, using CRISPR-Cas9, increased the expression of the MDM2-ALT1 homolog Mdm2-MS2. IMPLICATIONS: Taken together, the data indicate that modulating MDM2 splicing may be a useful tool for fine-tuning p53 activity in response to genotoxic stress.

Competitive ubiquitination activates the tumor suppressor p53

Blocking p53 ubiquitination through disrupting its interaction with MDM2 or inhibiting the MDM2 catalytic activity is the central mechanism by which the tumor suppressor p53 is activated in response to genotoxic challenges. Although MDM2 is first characterized as the major E3 ubiquitin ligase for p53, it can also catalyze the conjugation of ubiquitin moieties to other proteins (e.g., activating transcription factor 3, or ATF3). Here we report that ATF3 can act as an ubiquitin “trap” and competes with p53 for MDM2-mediated ubiquitination. While ATF3-mediated p53 stabilization required ATF3 binding to the MDM2 RING domain, we demonstrated that ATF3 ubiquitination catalyzed by MDM2 was indispensable for p53 activation in response to DNA damage. Moreover, a cancer-derived ATF3 mutant (R88G) devoid of ubiquitination failed to prevent p53 from MDM2-mediated degradation and thus was unable to activate the tumor suppressor. Therefore, we have identified a previously-unknown mechanism that can activate p53 in the genotoxic response.

Abstract P2018: PRR Mediates P53 Activation in Aristolochic Acid Induced Renal Proximal Tubule Cell Injury

Aristolochic acid (AA) induces renal proximal tubular injury leading to inflammation, atrophy and fibrosis. These effects of AA were reported to be mediated by p53 induced cell cycle arrest and apoptosis. Recently, our laboratory demonstrated that the (pro)renin receptor (PRR) promotes podocyte inflammation and apoptosis. We hypothesized that PRR mediates AA induced proximal tubular injury via upregulation of p53 activation. Mouse immortalized proximal tubule cells (PTCs) were grown to 70% confluence and transfected with either scramble (Scr) or PRR siRNA followed by treatment with either AA 10ug/mL or culture media alone (control) for 48 hours prior to harvesting for RNA and protein extraction (n=3 for all treatment groups). Compared to control, AA treatment alone significantly increased PRR mRNA expression by 202% (p<0.05). Compared to Scr treatment, PRR siRNA reduced PRR mRNA expression by 49% (p<0.05). Compared to AA treatment, AA+PRR siRNA ameliorated the observed increase in PRR mRNA. Total p53 mRNA expression was not significantly changed by treatment with PRR siRNA or AA. However, compared to the control group, p53 phosphorylation (activity) increased by 167% in the AA treated group (p<0.05). PRR shRNA attenuated (40% reduction) the observed increase in p53 phosphorylation that was induced by AA treatment (p<0.05). We conclude that PRR mediates increased activity of p53 in response to AA in proximal tubule cells.

The crystal structure of the protein kinase HIPK2 reveals a unique architecture of its CMGC-insert region

The homeodomain-interacting protein kinase (HIPK) family is comprised of four nuclear protein kinases, HIPK1-4. HIPK proteins phosphorylate a diverse range of transcription factors involved in cell proliferation, differentiation, and apoptosis. HIPK2, thus far the best-characterized member of this largely understudied family of protein kinases, plays a role in the activation of p53 in response to DNA damage. Despite this tumor-suppressor function, HIPK2 is also found overexpressed in several cancers, and its hyperactivation causes chronic fibrosis. There are currently no structures of HIPK2 or of any other HIPK kinase. Here, we report the crystal structure of HIPK2's kinase domain bound to CX-4945, a casein kinase 2α (CK2α) inhibitor currently in clinical trials against several cancers. The structure, determined at 2.2 Å resolution, revealed that CX-4945 engages the HIPK2 active site in a hybrid binding mode between that seen in structures of CK2α and Pim1 kinases. The HIPK2 kinase domain crystallized in the active conformation, which was stabilized by phosphorylation of the activation loop. We noted that the overall kinase domain fold of HIPK2 closely resembles that of evolutionarily related dual-specificity tyrosine-regulated kinases (DYRKs). Most significant structural differences between HIPK2 and DYRKs included an absence of the regulatory N-terminal domain and a unique conformation of the CMGC-insert region and of a newly defined insert segment in the αC-β4 loop. This first crystal structure of HIPK2 paves the way for characterizing the understudied members of the HIPK family and for developing HIPK2-directed therapies for managing cancer and fibrosis.

Abstract A23: Mdm2 phosphorylation by Akt is critical for modulating cellular responses to oxidative stress

Abstract The serine/threonine kinase Akt is activated by a myriad of growth signals and promotes cell proliferation, survival, and metabolism. Akt is frequently activated in a wide assortment of human hematologic malignancies and solid tumors. In vitro studies demonstrated that Akt phosphorylates the Mdm2 oncoprotein at ser166 and ser186 (163 and 183 in mice) to promote Mdm2 translocation into the nucleus. This enhances Mdm2-mediated p53 ubiquitination and the subsequent proteasomal-mediated degradation of p53. Wild-type p53 protein is a tumor suppressor that is activated in response to stress signals such as DNA damage, oxidative stress, and aberrant oncogene activation. Activated p53 induces the expression of genes that trigger cell cycle arrest, senescence, and apoptosis. This inhibits the growth of damaged and/or potentially oncogenic cells, underscoring the role of p53 in tumor suppression. However, the role of Akt-mediated phosphorylation of Mdm2 on p53 functions in vivo remains unknown. We hypothesize that Mdm2 phosphorylation by Akt at ser183 promotes cell growth and tumorigenesis by reducing p53 levels and function in vivo. Using CRISPR/Cas9-mediated gene editing, we generated mice with mutant Mdm2 protein that cannot be selectively phosphorylated at ser183, by substituting this serine with an alanine. Unlike Mdm2-/- mice that are embryonic lethal, Mdm2S183A mice are viable, fertile, and obtained at Mendelian ratios. There are no apparent growth/weight differences between unchallenged Mdm2WT and Mdm2S183A mice. Other stress-induced kinases like ATM and c-Abl phosphorylate Mdm2 to regulate the levels and functional activity of p53 in response to stress such as DNA damage. In contrast, Akt-mediated phosphorylation of Mdm2 does not alter p53 levels and function in response to DNA damage both in vitro and in vivo. However, under low-density stress plating, the ability of Mdm2S183A MEFs to form colonies is completely abrogated. In addition, Mdm2S183A MEFs fail to proliferate under normal cell culture conditions and exhibit salient features of senescence including positive staining for SA-β Gal. The expression of p53-target genes that mediate senescence is elevated in Mdm2S183A MEFs than in Mdm2WT MEFs. The severe proliferation defect and premature senescent phenotypes observed in Mdm2S183A MEFs are rescued by culturing cells at low oxygen tension with media supplemented with N-acetyl cysteine, a reactive oxygen species (ROS) scavenger. Our results from various assays examining the levels and effects of ROS suggest that under normal cell culture conditions, Mdm2S183A MEFs are more sensitive to oxidative stress compared to Mdm2WT MEFs. So far, our data suggest that Mdm2 phosphorylation by Akt at ser183 is required for proper regulation of the oxidative stress response and is necessary to promote cell growth under normal plating stress conditions. We are currently analyzing RNA-seq data to determine whether there are certain gene expression changes that could aid our quest to decipher the underlying mechanism responsible for the senescent phenotype of Mdm2S183A MEFs. In addition, we have crossed Mdm2S183A to lung and liver cancer models in which ROS plays a pivotal role to examine whether blocking Mdm2 phosphorylation by Akt alters the incidence and progression of tumors. Citation Format: Loretah Chibaya, Hong Zhang, Stephen N. Jones. Mdm2 phosphorylation by Akt is critical for modulating cellular responses to oxidative stress [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A23.

Nuclear retention of the lncRNA SNHG1 by doxorubicin attenuates hnRNPC-p53 protein interactions.

The protein p53 plays a crucial role in the regulation of cellular responses to diverse stresses. Thus, a major priority in cell biology is to define the mechanisms that regulate p53 activity in response to stresses or maintain it at basal levels under normal conditions. Moreover, further investigation is required to establish whether RNA participates in regulating p53's interaction with other proteins. Here, by conducting systematic experiments, we discovered a p53 interactor-hnRNPC-that directly binds to p53, destabilizes it, and prevents its activation under normal conditions. Upon doxorubicin treatment, the lncRNA SNHG1 is retained in the nucleus through its binding with nucleolin and it competes with p53 for hnRNPC binding, which upregulates p53 levels and promotes p53-dependent apoptosis by impairing hnRNPC regulation of p53 activity. Our results indicate that a balance between lncRNA SNHG1 and hnRNPC regulates p53 activity and p53-dependent apoptosis upon doxorubicin treatment, and further indicate that a change in lncRNA subcellular localization under specific circumstances is biologically significant.

Downregulation of Mir-146a-5p, Mir-99b-5p, Mir-143-3p, Mir-10a-5p and Mir-151a-3p Associated with PI3K/AKT, p53, NF-Kb, and Fanconi Anemia/BRCA Signaling Pathways Are Observed in Imatinib-Resistant Chronic Myeloid Leukemia Patients without Detectable BCR-ABL kinase Domain Mutations

IntroductionChronic myeloid leukemia (CML) patients who do not achieve landmark responses are considered imatinib (IM)-resistant. IM-resistance can be due to BCR-ABL kinase domain (KD) mutations, although many IM-resistant patients do not harbor KD mutations. The pathogenesis of this phenomenon is unclear. Aberrations in BCR-ABL independent downstream signaling pathways, namely PI3K/AKT, p53, NF-kB and Fanconi anaemia (FA)/BRCA, have been implicated. MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression and are notoriously promiscuous, with one miRNA regulating many mRNAs. In recognition of this, we sought to identify dysregulated miRNAs associated with the above pathways in patients with IM-resistant CML.MethodsEight patients with chronic phase CML who demonstrated primary resistance to IM and tested negative for KD mutations via Sanger sequencing were enrolled. Two healthy volunteers constituted the control group. Peripheral blood samples were taken and miRNA extracted from the white cells. MiRNA profiling was performed using miRNA sequencing (miRNA-seq) of the human microRNAome. MiRNA expression analyses (miEA) were performed on 4 sets of miRNAs associated with 4 different signaling pathways derived from publicly available databases: 237 miRNAs for PI3K/AKT, 223 miRNAs for p53, 221 miRNAs for NF-kB, and 126 miRNAs for FA/BRCA. MiRNA expression levels of the patients were compared with normal controls to obtain the differential expression of the miRNAs. MiRNAs that occur in all 4 pathways were further investigated in silico for their putative gene targets (Tarbase v7.0). Gene targets which were commonly linked with 3 different miRNAs were identified.ResultsMiRNA-seq yielded a total of 790 miRNAs. MiEA showed downregulation of 5 miRNAs in all 4 signaling pathways above: miR-146a-5p, miR-99b-5p, miR-143-3p and miR-10a-5p, miR-151a-3p. MiR-146a-5p and miR-99b-5p were the first and second most downregulated miRNAs in all 4 pathways. Putative gene targets that were commonly modulated by 3 miRNAs were: CDK6 (miR-143-3p, miR-10a-5p, miR-151a-3p), MDM2 (miR-146a-5p, miR-143-3p, miR-10a-5p), PTGS2 (miR-146a-5p, miR-143-3p, miR-10a-5p) and REV3L (miR-146a-5p, miR-143-3p, miR-151a-3p), as shown in Table 1.ConclusionsThe PI3K/AKT pathway is activated in response to IM-exposure to IM-naïve cells, implying a role in IM resistance. As cell cycle modulators, both p53 and NF-kB pathways are regulated within the PI3K/AKT pathway via AKT. FANCD2 is a central FA/BRCA pathway regulator and its transcription is maintained by AKT/mTOR via enhancement of NF-kB activity. The complexity of the interactions above has impeded efforts in identifying a unifying target in counteracting IM resistance. Thus, it is of significance that our results showed 5 differentially expressed miRNAs that consistently featured in the above signaling pathways, 4 of which remarkably influence PI3K/AKT and p53.In our study, PI3K/AKT and p53 pathways are inextricably linked with the same 4 miRNAs (miR-146a-5p, miR-143-3p, miR-10a-5p and miR-151a-3p) targeting the same 2 genes (MDM2 and CDK6) in these 2 pathways. MDM2 is an ubiquitin ligase that regulates the stability of p53a. AKT facilitates the functions of MDM2 to promote p53 ubiquitination. Conversely, inhibition of MDM2 leads to activation of p53 in response to cellular stresses. CDK6 is a cell cycle regulator and its overexpression influences the accumulation of pro-apoptotic p53 proteins, postulating a role in DNA damage response. Both PTGS2 and REV3L genes were commonly targeted by 3 miRNAs, within the NF-kB and FA/BRCA pathways respectively. PTGS2 overexpression has been shown in gastrointestinal adenocarcinomas; it induces carcinoma cell migration and invasion via activation of PI3K/AKT signaling. REV3L is the catalytic subunit of DNA polymerase ζ and plays a central role in DNA damage tolerance and chemoresistance towards DNA damaging agents. Notably, the NF-kB and FA/BRCA pathways were juxtaposed to the PI3K/AKT/p53 system via shared miRNA regulators.This provokes interesting questions regarding IM-resistance: are anti-apoptotic signaling and DNA damage response mechanisms driven simultaneously by these miRNAs? Can these miRNAs be potential biomarkers and therapeutic targets in IM-resistant CML? More studies are needed to further validate our findings. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chromosome missegregation during anaphase triggersp53 cell cycle arrest through histone H3.3 Ser31phosphorylation.

Maloriented chromosomes can evade the spindle assembly checkpoint and generate aneuploidy, a common feature of tumorigenesis. But chromosome missegregation in non-transformed cells triggers a p53-dependent fail-safe mechanism that blocks proliferation of normal cells that inadvertently become aneuploid. How this fail-safe is triggered is not known. Here we identify a conserved feedback mechanism that monitors missegregating chromosomes during anaphase through the differential phosphorylation of histone H3.3 at Ser31. We do this by inducing transient chromosome missegregation in diploid cells. During anaphase, H3.3 Ser31 is phosphorylated along the arms of lagging or misaligned chromosomes. Within minutes, Ser31 phosphorylation (Ser31P) spreads to all of the chromatids of both daughter cells, which persists into G1. Masking H3.3 Ser31P by antibody microinjection prevents nuclear p53 accumulation in the aneuploid daughters. Previous work demonstrated that prolonged prometaphase and DNA damage during abnormal mitosis can activate p53. We show that p53 activation in response to chromosome missegregation can occur without prolonged mitosis or DNA damage. Our study provides insight into how aneuploidy caused by chromosome missegregation is normally monitored and suppressed.

PIM1 destabilization activates a p53-dependent response to ribosomal stress in cancer cells.

Defects in ribosome biogenesis triggers a stress response (ribosomal stress) that can lead to growth arrest and apoptosis. Signaling pathways activated by ribosomal stress are specifically involved in the pathological mechanism of a group of disorders defined as ribosomopathies. However, more generally, the quality control of ribosome synthesis is part of the regulatory circuits that control cell metabolism. A number of studies identified tumor suppressor p53 as a central player in ribosomal stress. We have previously reported that the kinase PIM1 plays a role as a sensor for ribosome deficiency. In this report we address the relationship between PIM1 and p53 in cancer cell lines after depletion of a ribosomal protein. We identified a novel signaling pathway that includes the kinase AKT and the ubiquitin ligase MDM2. In fact, our results indicate that the lower level of PIM1, induced by ribosomal stress, causes inactivation of AKT, inhibition of MDM2 and a consequent p53 stabilization. Therefore, we propose that activation of p53 in response to ribosomal stress, is dependent on the pathway PIM1-AKT-MDM2. In addition, we report evidence that PIM1 level may be relevant to assess the sensitivity of cancer cells to chemotherapeutic drugs that induce ribosomal stress.

Mitochondrial dysfunction induces SESN2 gene expression through Activating Transcription Factor 4

ABSTRACTWe found that inhibitors of mitochondrial respiratory chain complexes III (myxothiazol) and I (piericidin A) in some epithelial carcinoma cell lines induce transcription of the p53-responsive SESN2 gene that plays an important role in stress response and homeostatic regulation. However, the effect did not depend on p53 because i) there was no induction of p53 after the treatment with piericidin A; ii) after the treatment with myxothiazol the peak of SESN2 gene upregulation occurred as early as 5h, before the onset of p53 activation (13h); iii) a supplementation with uridine that abolishes the p53 activation in response to myxothiazol did not abrogate the induction of SESN2 transcripts; iv) in the p53 negative HCT116 p53 -/- cells SESN2 transcription could be also induced by myxothiazol. In response to the respiratory chain inhibitors we observed an induction of ATF4, the key transcription factor of the integrated stress response (ISR). We found that the induction of SESN2 transcripts could be prevented by the ISR inhibitory small molecule ISRIB. Also, by inhibiting or overexpressing ATF4 with specific shRNA or ATF4-expressing constructs, respectively, we have confirmed the role of ATF4 in the SESN2 gene upregulation induced by mitochondrial dysfunction. At a distance of 228 bp upstream from the SESN2 transcription start site we found a candidate sequence for the ATF4 binding site and confirmed its requirement for the induction of SESN2 in luciferase reporter experiments. We suggest that the upregulation of SESN2 by mitochondrial dysfunction provides a homeostatic feedback that attenuates biosynthetic processes during temporal losses of energy supply from mitochondria thereby assisting better adaptation and viability of cells in hostile environments.