P53 Deficiency Research Articles

Role of p53 in the chronic pulmonary immune response to tangled or rod-like multi-walled carbon nanotubes

The fiber-like shape of multi-walled carbon nanotubes (MWCNTs) is reminiscent of asbestos, suggesting they pose similar health hazards when inhaled, including pulmonary fibrosis and mesothelioma. Mice deficient in the tumor suppressor p53 are susceptible to carcinogenesis. However, the chronic pathologic effect of MWCNTs delivered to the lungs of p53 heterozygous (p53+/−) mice has not been investigated. We hypothesized that p53+/− mice would be susceptible to lung tumor development after exposure to either tangled (t-) or rod-like (r-) MWCNTs. Wild-type (p53+/+) or p53+/− mice were exposed to MWCNTs (1 mg/kg) via oropharyngeal aspiration weekly over four consecutive weeks and evaluated for cellular and pathologic outcomes 11-months post-initial exposure. No lung or pleural tumors were observed in p53+/+ or p53+/− mice exposed to either t- or rMWCNTs. In comparison to tMWCNTs, the rMWCNTs induced the formation of larger granulomas, a greater number of lymphoid aggregates and greater epithelial cell hyperplasia in terminal bronchioles in both p53+/− and p53+/+ mice. A constitutively larger area of CD45R+/CD3+ lymphoid tissue was observed in p53+/− mice compared to p53+/+ mice. Importantly, p53+/− mice had larger granulomas induced by rMWCNTs as compared to p53+/+ mice. These findings indicate that a combination of p53 deficiency and physicochemical characteristics including nanotube geometry are factors in susceptibility to MWCNT-induced lymphoid infiltration and granuloma formation.

Abnormal gametogenesis induced by p53 deficiency promotes tumor progression and drug resistance

The century-old embryonal/gametogenesis hypothesis of tumors could link diverse tumors’ malignant features together likely representing the real “stemness” of tumors. However, the genetic evidence to validate abnormal gametogenesis in tumors remains lacking. Here we show that p53 deficiency elicits abnormal gametogenesis from primordial germ cell-like stage to late oocyte-like stage and subsequent parthenogenetic activation. The similar upregulation of abnormal gametogenesis by p53 deficiency is observed both in p53−/− mouse model and cultured cancer cells. Notably, germ cell-like cells isolated from distinct tumors from p53−/− mice and cancer cell lines display potent tumorigenicity potential. Abnormal oogenesis induced by p53 deficiency and then spontaneous parthenogenetic activation endow tumors with imitated embryonic development, life cycle, and therapeutic resistance. Our study establishes the genetic evidence to support embryonal/gametogenesis theory of tumors and reveals a pivotal role of p53 in restricting abnormal gametogenesis that may represent a novel aspect for p53’s tumor suppression.

Genetic deficiency of the tumor suppressor protein p53 influences erythrocyte survival.

The transcription factor p53 suppresses tumor growth by inducing nucleated cell apoptosis and cycle arrest. Because of its influence on primitive erythroid cell differentiation and survival, p53 is an important determinant of erythropoiesis. However, the impact of p53 on the fate of erythrocytes, cells lacking nucleus and mitochondria, during their post-maturation phase in the circulation remained elusive. Erythrocyte survival may be compromised by suicidal erythrocyte death or eryptosis, which is hallmarked by phosphatidylserine translocation and stimulated by increase of cytosolic Ca2+ concentration. Here, we comparatively examined erythrocyte homeostasis in p53-mutant mice (Trp53tm1Tyj/J) and in corresponding WT mice (C57BL/6J) by analyzing eryptosis and erythropoiesis. To this end, spontaneous cell membrane phosphatidylserine exposure and cytosolic Ca2+ concentration were higher in erythrocytes drawn from Trp53tm1Tyj/J mice than from WT mice. Eryptosis induced by glucose deprivation, a pathophysiological cell stressor, was slightly, but significantly more prominent in erythrocytes drawn from Trp53tm1Tyj/J mice as compared to WT mice. The loss of erythrocytes by eryptosis was fully compensated by enhanced erythropoiesis in Trp53tm1Tyj/J mice, as reflected by increased reticulocytosis and abundance of erythroid precursor cells in the bone marrow. Accordingly, erythrocyte number, packed cell volume and hemoglobin were similar in Trp53tm1Tyj/J and WT mice. Taken together, functional p53 deficiency enhances the turnover of circulating erythrocytes by parallel increase of eryptosis and stimulated compensatory erythropoiesis.

Human Serum Albumin and the p53-Derived Peptide Fusion Protein Promotes Cytotoxicity Irrespective of p53 Status in Cancer Cells.

Human serum albumin (HSA) fusion protein is a viable and effective approach to target and inhibit essential intracellular pathways. It has previously been shown that an HSA fusion protein containing a p53-reactivating peptide (rHSA-p53i) retains the binding activity to MDM2 and MDMX, resulting in p53 transcription-dependent apoptosis. Here, we demonstrate that rHSA-p53i is able to bind and neutralize anti-apoptotic Bcl-2 family proteins, Bcl-xL and Mcl-1. This interaction displaces pro-apoptotic Bak and subsequently leads to intrinsic apoptosis via mimicking a p53 transcription-independent pathway. Cytotoxicity induced by rHSA-p53i, via p53 transcription dependent and independent apoptotic pathways, is irrespective of the p53 status in MDA-MB-231, HeLa, and SJSA-1 cells possessing either mutant, deficient, or wild-type p53. The therapeutic potential is also confirmed by treating SJSA-1 and MDA-MB-231 xenograft mouse tumors with rHSA-p53i. These data reveal that rHSA-p53i interferes with at least four intracellular targets, making it a viable therapeutic protein for the treatment of a variety of cancers, as well as a carrier to deliver fatty acid-modified chemotherapeutics.

High-throughput gene expression analysis identifies p53-dependent and -independent pathways contributing to the adrenocortical dysplasia (acd) phenotype

In mammalian cells TPP1, encoded by the Acd gene, is a key component of the shelterin complex, which is required for telomere length maintenance and telomere protection. In mice, a hypomorphic mutation in Acd causes the adrenocortical dysplasia (acd) phenotype, which includes limb and body axis anomalies, and perinatal lethality. p53 deficiency partially rescues limb and body axis anomalies in acd mutant embryos, but not perinatal lethality, implicating p53-independent mechanisms in the acd phenotype. Loss of function of most shelterin proteins results in early embryonic lethality. Thus, study of the hypomorphic acd allele provides a unique opportunity to understand telomere dysfunction at an organismal level. The aim of this study was to identify transcriptome alterations in acd mutant and acd, p53 double mutant embryos to understand the p53-dependent and –independent factors that contribute to the mutant phenotypes in the context of the whole organism. Genes involved in developmental processes, cell cycle, metabolic pathways, tight junctions, axon guidance and signaling pathways were regulated by p53-driven mechanisms in acd mutant embryos, while genes functioning in immune response, and RNA processing were altered independently of p53 in acd, p53 double mutant embryos. To our best of knowledge, this is the first study revealing detailed transcriptomic alterations, reflecting novel p53-dependent and -independent pathways contributing to the acd phenotype. Our data confirm the importance of cell cycle and DNA repair pathways, and suggest novel links between telomere dysfunction and immune system regulation and the splicing machinery. Given the broad applicability of telomere maintenance in growth, development, and genome stability, our data will also provide a rich resource for others studying telomere maintenance and DNA damage responses in mammalian model systems.

Replication stress response in cancer stem cells as a target for chemotherapy.

Cancer stem cells (CSCs) are subpopulations of multipotent stem cells (SCs) responsible for the initiation, long-term clonal maintenance, growth and spreading of most human neoplasms. Reportedly, CSCs share a very robust DNA damage response (DDR) with embryonic and adult SCs, which allows them to survive endogenous and exogenous genotoxins. A range of experimental evidence indicates that CSCs have high but heterogeneous levels of replication stress (RS), arising from, and being boosted by, endogenous causes, such as specific genetic backgrounds (e.g., p53 deficiency) and/or aberrant karyotypes (e.g., supernumerary chromosomes). A multipronged RS response (RSR) is put in place by CSCs to limit and ensure tolerability to RS. The characteristics of such dedicated cascade have two opposite consequences, both relevant for cancer therapy. On the one hand, RSR efficiency often increases the reliance of CSCs on specific DDR components. On the other hand, the functional redundancy of pathways of the RSR can paradoxically promote the acquisition of resistance to RS- and/or DNA damage-inducing agents. Here, we provide an overview of the molecular mechanisms of the RSR in cancer cells and CSCs, focusing on the role of CHK1 and some emerging players, such as PARP1 and components of the homologous recombination repair, whose targeting can represent a long-term effective anti-CSC strategy.

Triptolide induces p53-dependent cardiotoxicity through mitochondrial membrane permeabilization in cardiomyocytes

Triptolide (TP), a major active component of Tripterygium wilfordii Hook f., is widely used in the treatment of inflammation and autoimmune disorders. Its clinical application is limited by severe adverse effects, especially cardiotoxicity. Accumulative evidences indicate that TP induces DNA damage by inhibiting RNA polymerase. Considering the relationship among DNA damage, p53, and the role of p53 in mitochondria-dependent apoptosis, we speculate that TP-induced cardiotoxicity results from p53 activation. In this study, the role of p53 in TP-induced cardiotoxicity was investigated in H9c2 cells, primary cardiomyocytes, and C57BL/6 genetic background p53−/− mice. p53 protein level was elevated by TP in vitro and in acute heart injury models. With TP administration (1.2 mg/kg), p53 deficiency prevented heart histology injury and decreased serum cardiac troponin I (cTn-I) and apoptotic proteins. Mechanistically, immunoblotting and immunofluorescence staining identified that TP-induced toxicity is dependent on p53 nuclear translocation and transactivation of Bcl2 family genes, leading to mitochondrial outer membrane permeabilization (MOMP) and mitochondria dysfunction. Consistently, p53 antagonist PFTα counteracted TP-induced p53 overexpression and regulation of Bcl2 family transcription, which improved mitochondrial membrane integrity and prevented apoptosis. Moreover, Bax antagonist Bax inhibitor peptide (BIP) V5 ameliorated TP-induced apoptosis through suppressing membrane depolarization and ROS accumulation. These results suggest that TP-induced cardiotoxicity is p53-dependent by promoting Bax-induced mitochondria-mediated apoptosis.

Generation of Immortalised But Unstable Cells after hTERT Introduction in Telomere-Compromised and p53-Deficient vHMECs.

Telomeres, the natural ends of chromosomes, hide the linear telomeric DNA from constitutive exposure to the DNA damage response with a lariat structure or t-loop. Progressive telomere shortening associated with DNA replication in the absence of a compensatory mechanism culminates in t-loop collapse and unmasked telomeres. Dysfunctional telomeres can suppress cancer development by engaging replicative senescence or apoptosis, but they can also promote tumour initiation when cell cycle checkpoints are disabled. In this setting, telomere dysfunction promotes increasing chromosome instability (CIN) through breakage-fusion-bridge cycles. Excessive instability may hamper cell proliferation but might allow for the appearance of some rare advantageous mutations that could be selected and ultimately favour neoplastic progression. With the aim of generating pre-malignant immortalised cells, we ectopically expressed telomerase in telomere-compromised variant human mammary epithelial cells (vHMECs), proficient and deficient for p53, and analysed structural and numerical chromosomal aberrations as well as abnormal nuclear morphologies. Importantly, this study provides evidence that while immortalisation of vHMECs at early stages results in an almost stable karyotype, a transient telomere-dependent CIN period—aggravated by p53 deficiency—and followed by hTERT overexpression serves as a mechanism for the generation of immortal unstable cells which, due to their evolving karyotype, could attain additional promoting properties permissive to malignancy.

PO-288 Replication stress response as a target for eradicating colorectal cancer stem cells

IntroductionCancer stem cells (CSCs) are subsets of multipotent SCs responsible for tumour development, propagation and evolution, whose targeting is required for tumour eradication. There is (pre)clinical evidence on a role of CSCs in therapeutic resistance and intra-tumour heterogeneity, which limits the efficacy of antineoplastic regimens. In this context, CSCs reportedly share with embryonic/adult SCs a very robust DNA damage response, which favours the survival and resistance to genotoxins, and can be exploited for therapeutic purposes.Material and methodsWe generated a panel of ~30 CRC patient-derived tumorspheres enriched for CSCs (CRC-SCs) and characterised them at the genetic level. To discover potential monotherapeutic anti-CSC agents, we performed high-throughput screenings on multiple CRC-SCs with a library of clinically-relevant drugs. Flow cytometry, fluorescence microscopy and epistatic analyses were conducted to uncover the mechanism of action of identified compound(s), while genetic, cytogenetic and phosphoproteomic studies were carried out to identify predictive biomarkers. DNA replication stress (RS) levels were evaluated by analysing phosphorylated ATM/RPA foci and by performing COMET and DNA fibre assays, and were modulated by single administration of genome destabilising agents or by prolonged exposure to increased doses of compounds targeting the replication stress response (RSR).Results and discussionsWe demonstrated that the CHK1 inhibitor LY2606368 is a potent anti-CSC agent able to kill more than one third of CRC-SCs, both in vitro and in vivo. Moreover, we provided evidence of high but heterogeneous RS levels in CRC-SCs, showing that, in CRC-SCs, RS is mainly boosted endogenously by p53 deficiency, supernumerary chromosomes and DNA replication abnormalities, which results in high dependency on CHK1-mediated RSR. Accordingly, formerly LY2606368-resistant CRC-SCs were sensitised by boosting DNA replication errors or inducing whole-genome doubling, while formerly LY2606368-sensitive CRC-SCs made resistant by the continuous in vitro or in vivo administration of LY2606368 displayed diminished RS levels due to RSR rewiring, and became independent on CHK1.ConclusionOur results demonstrate that RSR is efficient and rewirable in CSCs thereby constituting a prominent therapeutic target. In particular, we designed dedicated RS-modulating or RSR-targeting strategies for long-term CSC depletion in CRC.

Abstract 4867: Treating hepatocellular carcinoma metastasis and overcoming chemoresistance through inhibiting the MDM2 oncogene

Abstract Hepatocellular carcinoma (HCC) poses a major health problem worldwide and novel, effective therapeutic approaches are urgently needed. The MDM2 oncogene is amplified and overexpressed in HCC and associated with its initiation, progression, metastasis, and chemoresistence. MDM2 has been demonstrated to be as a promising therapeutic target for the treatment of HCC. However, most of the current MDM2 inhibitors have been designed to block the binding between MDM2 and p53, and have limited efficacy against tumors with mutant or deficient p53. We recently performed a high-throughput virtual and cell-based screening, yielding a novel MDM2 inhibitor, termed SP141. The present study was designed to evaluate the anti-HCC activity of SP141, its potential to chemosensitize HCC cells to other anticancer agents, and to determine the underlying mechanism(s) of action of SP141 in various in vitro and in vivo models with different backgrounds of p53 (wildtype, mutant or null). Our results demonstrated that SP141 inhibited cell growth and prevented cell migration and invasion, independent of p53. Mechanistically, SP141 directly bound to the MDM2 protein and promoted MDM2 degradation. The inhibition of MDM2 by SP141 also increased the sensitivity of HCC cells to sorafenib. In addition, in orthotopic and patient-derived xenograft (PDX) models, SP141 inhibited MDM2 expression and suppressed tumor growth and metastasis, without any host toxicity. Furthermore, mechanistic study demonstrated that the inhibition of MDM2 by SP141 was essential for its anti-HCC activities. In conclusion, MDM2 inhibition by SP141 resulted in the inhibition of HCC tumor growth and metastasis and sensitization of HCC cells to chemotherapy, regardless of their p53 status. These results provide support for the further development of SP141 as a lead candidate for the treatment of HCC. (Supported by NIH R01 CA186662 and R01CA214019 and ACS RSG-15-009-01-CDD.) Citation Format: Wei Wang, Bo Hu, Jiang-Jiang Qin, Jianwen Cheng, Xin Li, Ming Hu, John Buolamwini, Xin-Rong Yang, Jia Fan, Ruiwen Zhang. Treating hepatocellular carcinoma metastasis and overcoming chemoresistance through inhibiting the MDM2 oncogene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4867.

Acrolein induces ribotoxic stress in human cancer cells regardless of p53 status

Acrolein (Acr) cytotoxicity contributes to chemotherapeutic activity of cyclophosphamide via metabolism of the anticancer drug. Our previous studies have shown that Acr causes ribosomal DNA (rDNA) damages, thus shuts down ribosomal RNA (rRNA) synthesis and leads to ribosomal stress in human cancer cells. Ribosome senses stress in 28S rRNA and induces subsequent activation of mitogen-activated protein kinase (MAPK) pathway which triggers ribotoxic stress response (RSR). Here, we report that cells harboring p53 or not responds differently to Acr-induced RSR. Our results show that Acr induced rRNA cleavage via the activated caspases in cancer cells with wild type p53, but not in cells with deficient p53. Furthermore, MAPK pathways were activated by Acr in cancer cells regardless of p53 status. Acr induced apoptosis in cells with wild type p53, while it induced G2/M cell cycle arrest in cancer cells with deficient p53. In conclusion, the presence of functional p53 plays a significant role in the mechanisms of Acr-induced rRNA cleavage and cell fates. Our results enhance our understanding of the molecular mechanisms of Acr-mediated antitumor activity which helps develop better therapeutic strategies for killing cancer cells with different p53 status.

CDKN1B/p27 is localized in mitochondria and improves respiration-dependent processes in the cardiovascular system-New mode of action for caffeine.

We show that the cyclin-dependent kinase inhibitor 1B (CDKN1B)/p27, previously known as a cell cycle inhibitor, is also localized within mitochondria. The migratory capacity of endothelial cells, which need intact mitochondria, is completely dependent on mitochondrial p27. Mitochondrial p27 improves mitochondrial membrane potential, increases adenosine triphosphate (ATP) content, and is required for the promigratory effect of caffeine. Domain mapping of p27 revealed that the N-terminus and C-terminus are required for those improvements. Further analysis of those regions revealed that the translocation of p27 into the mitochondria and its promigratory activity depend on serine 10 and threonine 187. In addition, mitochondrial p27 protects cardiomyocytes against apoptosis. Moreover, mitochondrial p27 is necessary and sufficient for cardiac myofibroblast differentiation. In addition, p27 deficiency and aging decrease respiration in heart mitochondria. Caffeine does not increase respiration in p27-deficient animals, whereas aged mice display improvement after 10 days of caffeine in drinking water. Moreover, caffeine induces transcriptome changes in a p27-dependent manner, affecting mostly genes relevant for mitochondrial processes. Caffeine also reduces infarct size after myocardial infarction in prediabetic mice and increases mitochondrial p27. Our data characterize mitochondrial p27 as a common denominator that improves mitochondria-dependent processes and define an increase in mitochondrial p27 as a new mode of action of caffeine.

PO-214 Conditional control of MITF reveals cellular subpopulations essential for melanoma survival and recurrence in new zebrafish models

Introduction Melanoma is the most lethal type of skin cancer and its incidence continues to rise rapidly. While BRAFV600E inhibitors have led to significant improvements in outcomes for some patients, there are no targeted therapies available for melanoma patients who lack BRAF mutations. An emerging area of new melanoma targets is the melanocyte lineage, which is critical for melanoma survival and contributes to therapeutic resistance. MITF is a conserved ‘master melanocyte regulator’ with a complex role in melanoma. Our lab previously developed a BRAFV600E mitf zebrafish melanoma model carrying a human oncogene and mitfvc7 splice site mutation that enables the conditional control of its endogenous activity. Since there are not many adequate animal models to study the non-BRAF and non-RAS melanomas, we have now also developed a double mutant model p53 mitf. Here we use the new models to investigate MITF-dependent melanoma regression and recurrence. Material and methods We use histopathology and imaging of melanocyte lineage fluorescent reporters (mitf and crestin) in new temperature-sensitive zebrafish models, as well as primary zebrafish melanoma cell line, to study melanoma regression and recurrence. Results and discussions We demonstrate that the MITF activity is crucial for melanocyte survival and that both mutated BRAF and p53 deficiency are oncogenic with low levels of MITF, and result in melanoma resembling the pathology of human disease. This is particularly important considering the new MITF-low subclass of human melanomas that has emerged from the recent TCGA classification. We also show that complete inhibition of MITF activity leads to rapid tumour regression (in both new models), but once its activity is restored the melanomas recur at the same site as the original tumour. This suggests that a subpopulation of cancer initiating cells remains following melanoma regression and is capable of repopulating the tumour. We have been able to show that some cells remaining at the sites of regression express MITF and/or ALDH2 and we are currently searching for other markers. Conclusion We developed new MITF-dependent zebrafish models of melanoma that are relevant to distinct clinical subtypes. We showed that inhibiting MITF in those animals results in melanoma regression, but is not sufficient to eradicate the tumours (once its activity is restored, melanomas recur). We identified subpopulations of cells remaining at the regression sites and are investigating the nature of these proposed cells-of-origin for relapse.

2213 Tumor suppressors p53 and ARF control oncogenic potential of triple-negative breast cancer cells by regulating RNA editing enzyme ADAR1

OBJECTIVES/SPECIFIC AIMS: Triple-negative breast cancer (TNBC) accounts for one-fifth of the breast cancer patient population. The heterogeneous nature of TNBC and lack of options for targeted therapy make its treatment a constant adventure. The deficiency of tumor suppressors p53 and ARF is one of the known genetic signatures enriched in TNBC. Crucial questions remain about how TNBC is regulated by these genetic alterations. METHODS/STUDY POPULATION: In order to address this issue, we established p53/ARF-defective murine embryonic fibroblast and mammary epithelial cell to study the molecular and phenotypic consequences. Moreover, transgenic mice were generated to investigate the effect of p53/ARF deficiency on mammary tumor development in vivo. RESULTS/ANTICIPATED RESULTS: Increased proliferation and transformation capability were observed in p53/ARF-defective cells, and an aggressive form of mammary tumor was also seen in p53−/−ARF−/− mice. Gene expression profiling and knock-down experiments using shRNAs were conducted to identify inflammatory marker ISG15 and RNA-editing enzyme ADAR1 as potential culprits for the elevated oncogenic potential. Interestingly, we found that the overexpression of ISG15 and ADAR1 is also prevalent in human TNBC cell lines. Reducing ADAR1 expression abrogated the oncogenic potential of human TNBC cell lines, while non-TNBC cells are less susceptible. DISCUSSION/SIGNIFICANCE OF IMPACT: These results indicate critical roles played by the tumor suppressors p53 and ARF in the pathogenesis of TNBC, likely through regulating ADAR1-mediated RNA modifications. Further understanding of this pathway promises to shed light on genetics-driven vulnerabilities of TNBC and inform development of more effective therapeutic strategies.

Flavan enantiomers from Daphne giraldii selectively induce apoptotic cell death in p53-null hepatocarcinoma cells in vitro

Hepatocellular carcinoma (HCC) is a primary malignant tumor with very poor prognosis. To search for more effective compounds for HCC treatment, two new pairs of flavan enantiomers, daphnegiranol C1/C2 (1a/1b) and daphnegiranol D1/D2 (2a/2b), were isolated from the stem bark and roots of Daphne giraldii by using chiral chromatography. MTT assay was applied to evaluate their cytotoxicity against three hepatocarcinoma cell lines (Hep3B, MHCC97H, HepG2) as well as a normal liver cell line L02. The results showed that these compounds preferred to inhibit the growth of Hep3B cells (p53 null). Among them, 2a/2b (the IC50 value was 4.87 and 3.35 μM, respectively) exhibited a stronger cytotoxic effect than sorafenib (IC50 = 6.59 μM) in Hep3B cells. A further study demonstrated that 2a/2b could induce apoptotic cell death with an increase in reactive oxygen species (ROS) in Hep3B cells. In addition, the IC50 values of 2a/2b in HepG2 and MHCC97H cells (both harbored p53 gene) were more than 10-folds greater when compared with Hep3B cells, indicating that 2a/2b selectively exhibited cytotoxicity in p53-null hepatocarcinoma cells. Moreover, inhibition of p53 increased the inhibitory effect in p53-wild hepatocarcinoma cells, as well as apoptotic cells and ROS generation. Taken together, flavan enantiomers 2a/2b selectively induced apoptosis in Hep3B cells because of p53 deficiency.

The human immune response to RSV includes a loop between the tumor suppressor p53 and expression of APOBEC3 genes

BackgroundRespiratory syncytial virus (RSV) infects nearly all children by theirsecond year of life with re‐infections occurring throughout adolescence andadulthood. RSV infection typicallyresults in mild host symptoms. However, susceptible individuals can develop severe lower respiratory tract infectionsand bronchiolitis, sometimes leading to hospitalization or death. Recently we established that in addition toits protective role against cancer development, the tumor suppressor p53 alsocontributes to host immune responses regulating the expression of several genesincluding the APOBEC3 family of DNA cytidine deaminases (A3), consideredsentinels in innate immune response against viral infections.ObjectiveUsinghuman lung cancer cells, we tested the role of p53 in response to RSV infectionand its replication through the regulation of the A3 gene family as well asother components of the immune response.MethodsA549 and H1299 cells were infected with RSV (Strain A2). Cells were thenharvested 72–120 hours post‐infection and qPCR and Nanostring arrays? were usedto evaluate mRNA expression of RSV‐g, P53, A3s and other immune targets. Expression of RSV was assessed? by ConfocalMicroscopy and cytofluorometry while Annexin V/PI assay was used to evaluateapoptosis.ResultsIn response to acute and chronic RSV infection in human lung cancer cells, p53 was activated and had an impact on RSV replication, which involves theexpression in a p53 dependent manner of several members of the A3 gene familyas well as other innate immune genes. APOBEC3G (A3G) was highly induced in a dose‐ and time‐dependent mannerafter RSV only in lung cancer cells harboring WT p53. Accordingly, overexpression of A3G in lungcancer cells reduced RSV replication. Furthermore, a Nanotstring transcriptome analysis (immune code set) after RSV infectionrevealed a strong p53 dependent regulation of immune response genes, includingseveral components of the innate immune antiviral response. In addition, RSV infection induced p53phosphorylation at serine 15 and triggered p53‐dependent apoptosis in p53 functional cells while p53 deficiency correlated with increased viralreplication and increased syncytia phenotype.ConclusionsOverall, our findings establish that the “guardian of the genome” role ascribed to p53 also extends to a unique component of the immune system – the A3 genes – revealing novel components in the antiviralimmune response against RSV infection.Support or Funding InformationSupported by NIEHS Division of Intramural Research Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P53-Mediated Molecular Control of Autophagy in Tumor Cells.

Autophagy is an indispensable mechanism of the eukaryotic cell, facilitating the removal and renewal of cellular components and thereby balancing the cell’s energy consumption and homeostasis. Deregulation of autophagy is now regarded as one of the characteristic key features contributing to the development of tumors. In recent years, the suppression of autophagy in combination with chemotherapeutic treatment has been approached as a novel therapy in cancer treatment. However, depending on the type of cancer and context, interference with the autophagic machinery can either promote or disrupt tumorigenesis. Therefore, disclosure of the major signaling pathways that regulate autophagy and control tumorigenesis is crucial. To date, several tumor suppressor proteins and oncogenes have emerged as eminent regulators of autophagy whose depletion or mutation favor tumor formation. The mammalian cell “janitor” p53 belongs to one of these tumor suppressors that are most commonly mutated in human tumors. Experimental evidence over the last decade convincingly reports that p53 can act as either an activator or an inhibitor of autophagy depending on its subcellular localization and its mode of action. This finding gains particular significance as p53 deficiency or mutant variants of p53 that accumulate in the cytoplasm of tumor cells enable activation of autophagy. Accordingly, we recently identified p53 as a molecular hub that regulates autophagy and apoptosis in histone deacetylase inhibitor-treated uterine sarcoma cells. In light of this novel experimental evidence, in this review, we focus on p53 signaling as a mediator of the autophagic pathway in tumor cells.

Inhibition of autophagy enhances DENSpm-induced apoptosis in human colon cancer cells in a p53 independent manner

One of the recently developed polyamine (PA) analogues, N 1 ,N11-diethylnorspermine (DENSpm), has been found to act as an apoptotic inducer in melanoma, breast, prostate and colon cancer cells. Also, its potential to induce autophagy has been established. Unfolded protein responses and starvation of amino acids are known to trigger autophagy. As yet, however, the molecular mechanism underlying PA deficiency-induced autophagy is not fully clarified. Here, we aimed to determine the apoptotic effect of DENSpm after autophagy inhibition by 3-methyladenine (3-MA) or siRNA-mediated Beclin-1 silencing in colon cancer cells. The apoptotic effects of DENSpm after 3-MA treatment or Beclin-1 silencing were determined by PI and AnnexinV/PI staining in conjunction with flow cytometry. Intracellular PA levels were measured by HPLC, whereas autophagy and the expression profiles of PA key players were determined in HCT116, SW480 and HT29 colon cancer cells by Western blotting. We found that DENSpm-induced autophagy was inhibited by 3-MA treatment and Beclin-1 silencing, and that apoptotic cell death was increased by PA depletion and spermidine/spermine N1-acetyltransferase (SSAT) upregulation. We also found that autophagy inhibition led to DENSpm-induced apoptosis through Atg5 down-regulation, p62 degradation and LC3 lipidation in both HCT116 and SW480 cells. p53 deficiency did not alter the response of the colon cancer cells to DENSpm-induced apoptotic cell death under autophagy suppression conditions. From our results we conclude that DENSpm-induced apoptotic cell death is increased when autophagy is inhibited by 3-MA or Beclin-1 siRNA through PA depletion and PA catabolic activation in colon cancer cells, regardless p53 mutation status.

Functional loss of p53 cooperates with the in vivo microenvironment to promote malignant progression of gastric cancers

p53 mutations are frequently detected in malignant gastric cancers. However, the molecular mechanisms by which loss of p53 function promotes gastric cancer are not clear. We utilized Gan mice (K19-Wnt1/C2mE), which have functional p53 and develop intestinal-type gastric tumors, to investigate the role of p53 in gastric cancer progression by knocking out p53. We found that gastric epithelial cells acquire tumorigenicity in the subcutis of C57BL/6 mice as a result of Wnt activation, COX-2 activation and p53 deficiency. With repeated allograft transfers, these gastric epithelial cells gradually acquired the properties of malignant gastric cancer. Loss of p53 conferred cell stemness and induced epithelial to mesenchymal transition (EMT) in gastric epithelial cells, and these properties were further enhanced by the in vivo microenvironment, ultimately leading to gastric cancer formation and metastasis. We also found that the in vivo microenvironment enhanced activation of the COX-2 pathway, which further contributed to cancer progression. With this system, we have succeeded in recapitulating the development of malignant gastric cancer from gastric epithelial cells in a normal immune environment.

Loss of Uracil DNA Glycosylase Selectively Resensitizes p53-Mutant and -Deficient Cells to 5-FdU.

Thymidylate synthase (TS) inhibitors including fluoropyrimidines [e.g., 5-Fluorouracil (5-FU) and 5-Fluorodeoxyuridine (5-FdU, floxuridine)] and antifolates (e.g., pemetrexed) are widely used against solid tumors. Previously, we reported that shRNA-mediated knockdown (KD) of uracil DNA glycosylase (UDG) sensitized cancer cells to 5-FdU. Because p53 has also been shown as a critical determinant of the sensitivity to TS inhibitors, we further interrogated 5-FdU cytotoxicity after UDG depletion with regard to p53 status. By analyzing a panel of human cancer cells with known p53 status, it was determined that p53-mutated or -deficient cells are highly resistant to 5-FdU. UDG depletion resensitizes 5-FdU in p53-mutant and -deficient cells, whereas p53 wild-type (WT) cells are not affected under similar conditions. Utilizing paired HCT116 p53 WT and p53 knockout (KO) cells, it was shown that loss of p53 improves cell survival after 5-FdU, and UDG depletion only significantly sensitizes p53 KO cells. This sensitization can also be recapitulated by UDG depletion in cells with p53 KD by shRNAs. In addition, sensitization is also observed with pemetrexed in p53 KO cells, but not with 5-FU, most likely due to RNA incorporation. Importantly, in p53 WT cells, the apoptosis pathway induced by 5-FdU is activated independent of UDG status. However, in p53 KO cells, apoptosis is compromised in UDG-expressing cells, but dramatically elevated in UDG-depleted cells. Collectively, these results provide evidence that loss of UDG catalyzes significant cell death signals only in cancer cells mutant or deficient in p53.Implications: This study reveals that UDG depletion restores sensitivity to TS inhibitors and has chemotherapeutic potential in the context of mutant or deficient p53. Mol Cancer Res; 16(2); 212-21. ©2017 AACR.