Absence Of P53 Research Articles

Endoderm development requires centrioles to restrain p53-mediated apoptosis in the absence of ERK activity.

Centrioles comprise the heart of centrosomes, microtubule-organizing centers. To study the function of centrioles in lung and gut development, we genetically disrupted centrioles throughout the mouse endoderm. Surprisingly, removing centrioles from the endoderm did not disrupt intestinal growth or development but blocked lung branching. In the lung, acentriolar SOX2-expressing airway epithelial cells apoptosed. Loss of centrioles activated p53, and removing p53 restored survival of SOX2-expressing cells, lung branching, and mouse viability. To investigate how endodermal p53 activation specifically killed acentriolar SOX2-expressing cells, we assessed ERK, a prosurvival cue. ERK was active throughout the intestine and in the distal lung buds, correlating with tolerance to centriole loss. Pharmacologically inhibiting ERK activated apoptosis in acentriolar cells, revealing that ERK activity protects acentriolar cells from apoptosis. Therefore, centrioles are largely dispensable for endodermal growth and the spatial distribution of ERK activity in the endoderm shapes the developmental consequences of centriolar defects and p53 activation.

Abstract PO-084: The role of p53 in the development of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a very deadly cancer, with a 5-year survival rate of only 9%. This high mortality rate can be attributed to the lack of early detection methods and to tumors being resistant to treatment, in part due to a highly immunosuppressive tumor microenvironment (TME). The most commonly mutated genes in PDAC include activating mutations in KRAS in >90% of cases and mutations in the tumor suppressor TP53 gene in ~72% of cases. However, how p53, which is a transcription factor, normally suppresses PDAC progression is not well understood. Understanding the pathways through which p53 acts to suppress PDAC will provide key insight into the molecular mechanisms underlying PDAC initiation and progression. Our laboratory has recently shown in genetically engineered mouse models that Kras activation, coupled with p53 inactivation, can drive PDAC from either pancreatic acinar or ductal cells, with acinar cell- and ductal cell-derived tumor signatures resembling the classical and basal-like human PDAC subtypes, respectively. To better understand how p53 loss promotes PDAC, we are focusing on the acinar cell-derived PDAC mouse model and using single-cell transcriptomic technologies to ask in which cell states and through which pathways p53 acts to suppress PDAC development, in particular as PDAC has complex interactions between tumor cells and the TME. In this model, PDAC is driven by oncogenic Kras (KrasG12D) along with either p53 wild type (p53wt) or p53 deficiency (p53flox) in adult acinar cells by a tamoxifen-inducible Ptf1aCreER allele. In the progression of this model, acinar cells can give rise to PDAC likely through a transdifferentiation process called acinar-to-ductal metaplasia (ADM), which leads to pancreatic intraepithelial neoplasias (PanINs) and then PDAC. The initial cell fate transition, ADM, can be activated by growth factors, inflammation, and injury. We have conducted single cell RNA-sequencing (scRNA-seq) to compare early stages of tumor development in the presence and absence of p53. We have also conducted spatial transcriptomic analysis of these samples. Using these combined approaches, we are analyzing tumor cell populations and cells of the tumor microenvironment in the p53wt and p53flox genotypes and have found. We will present our latest single cell analyses on p53 dependent cell states and functional pathways during PDAC development. Understanding how tumor cell states and cells of the tumor microenvironment change with p53 status will reveal different paths of PDAC evolution and uncover novel approaches to improve clinical intervention for human PDAC. Citation Format: Kathryn J. Hanson, Brittany M. Flowers, Nicholas Hughes, Hannes Vogel, Le Cong, Laura D. Attardi. The role of p53 in the development of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-084.

Shift in MSL1 alternative polyadenylation in response to DNA damage protects cancer cells from chemotherapeutic agent-induced apoptosis.

SUMMARYDNA damage reshapes the cellular transcriptome by modulating RNA transcription and processing. In cancer cells, these changes can alter the expression of genes in the immune surveillance and cell death pathways. Here, we investigate how DNA damage impacts alternative polyadenylation (APA) using the PAPERCLIP technique. We find that APA shifts are a coordinated response for hundreds of genes to DNA damage, and we identify PCF11 as an important contributor of DNA damage-induced APA shifts. One of these APA shifts results in upregulation of the full-length MSL1 mRNA isoform, which protects cells from DNA damage-induced apoptosis and promotes cell survival from DNA-damaging agents. Importantly, blocking MSL1 upregulation enhances cytotoxicity of chemotherapeutic agents even in the absence of p53 and overcomes chemoresistance. Our study demonstrates that characterizing adaptive APA shifts to DNA damage has therapeutic implications and reveals a link between PCF11, the MSL complex, and DNA damage-induced apoptosis.

Augmin deficiency in neural stem cells causes p53-dependent apoptosis and aborts brain development

Microtubules that assemble the mitotic spindle are generated by centrosomal nucleation, chromatin-mediated nucleation, and nucleation from the surface of other microtubules mediated by the augmin complex. Impairment of centrosomal nucleation in apical progenitors of the developing mouse brain induces p53-dependent apoptosis and causes non-lethal microcephaly. Whether disruption of non-centrosomal nucleation has similar effects is unclear. Here, we show, using mouse embryos, that conditional knockout of the augmin subunit Haus6 in apical progenitors led to spindle defects and mitotic delay. This triggered massive apoptosis and abortion of brain development. Co-deletion of Trp53 rescued cell death, but surviving progenitors failed to organize a pseudostratified epithelium, and brain development still failed. This could be explained by exacerbated mitotic errors and resulting chromosomal defects including increased DNA damage. Thus, in contrast to centrosomes, augmin is crucial for apical progenitor mitosis, and, even in the absence of p53, for progression of brain development.

WT1 inhibits AML cell proliferation in a p53-dependent manner

ABSTRACT WT1 has been reported to function as an oncogene and a tumor suppressor in acute myeloid leukemia (AML). The molecular mechanisms have not yet been fully elucidated. Here, we report that p53, served as a tumor suppressor, plays a critical role in regulating the function of WT1 in AML. For details, we performed a meta-analysis on 1131 AML cases, showing that WT1 gene mutation and TP53 gene exhibited a mutually exclusive predisposition in AML. p53 can be recruited to the promoter region of WT1’s target genes to modulate their expression by physically interacting with WT1. The AML-derived p53 mutation (p53R248Q) can disrupt the interaction between WT1 and p53, resulting in the loss of modulation of WT1’s target genes. Furthermore, wild-type p53 maintained the anti-proliferation activity of WT1 in AML cells. In contrast, WT1 promoted AML cell proliferation in the absence of p53 (or mutated p53). In conclusion, we demonstrated a novel explanation of the controversial function of WT1 in AML. These results provided a mechanism by which WT1 inhibited AML cell proliferation in a p53-dependent manner.

A Diarylpentanoid with Potential Activation of the p53 Pathway: Combination of in silico Screening Studies, Synthesis, and Biological Activity Evaluation.

In silico studies of a library of diarylpentanoids led us to the identification of potential new MDM2/X ligands. The diarylpentanoids with the best docking scores obeying the druglikeness and ADMET prediction properties were subsequently synthesized and evaluated for their antiproliferative activity on colon cancer HCT116 and fibroblasts HFF-1 cells. The effect on p53-MDM2/X interactions was evaluated through yeast-based assays for compounds showing potent antiproliferative activity in HCT116 cells and low toxicity in normal cells, resulting in the identification of a potential dual inhibitor. Moreover, its antiproliferative effect was significantly reduced in the absence of p53 and in MDA-MB-231 cells expressing a mutant p53 form. The antiproliferative effect of this compound was associated with induction of cell cycle arrest, apoptosis, PARP cleavage and increased p53 and its transcriptional targets, p21 and PUMA, in HCT116 cells. Docking poses and residues involved in the inhibition of p53-MDM2/X interactions were predicted by docking studies.

Transient genomic instability drives tumorigenesis through accelerated clonal evolution

Abnormal numerical and structural chromosome content is frequently found in human cancer. To test the role of aneuploidy in tumor initiation and progression, we generated mice with random aneuploidies by transient induction of polo-like kinase 4 (Plk4), a master regulator of centrosome number. Short-term chromosome instability (CIN) from transient Plk4 induction resulted in formation of aggressive T-cell lymphomas in mice with heterozygous inactivation of one p53 allele and accelerated tumor development in the absence of p53. Transient CIN increased the frequency of lymphoma-initiating cells with a specific karyotype profile, including trisomy of chromosomes 4, 5, 14, and 15 occurring early in tumorigenesis. Tumor development in mice with chronic CIN induced by an independent mechanism (through inactivation of the spindle assembly checkpoint) gradually trended toward a similar karyotypic profile, as determined by single-cell whole-genome DNA sequencing. Overall, we show how transient CIN generates cells with random aneuploidies from which ones that acquire a karyotype with specific chromosome gains are sufficient to drive cancer formation, and that distinct CIN mechanisms can lead to similar karyotypic cancer-causing outcomes.

Single loss of a Trp53 allele triggers an increased oxidative, DNA damage and cytokine inflammatory responses through deregulation of I\u03baB\u03b1 expression

Dose of Trp53, the main keeper of genome stability, influences tumorigenesis; however, the causes underlying and driving tumorigenesis over time by the loss of a single p53 allele are still poorly characterized. Here, we found that single p53 allele loss specifically impacted the oxidative, DNA damage and inflammatory status of hematopoietic lineages. In particular, single Trp53 allele loss in mice triggered oxidative stress in peripheral blood granulocytes and spleenocytes, whereas lack of two Trp53 alleles produced enhanced oxidative stress in thymus cells, resulting in a higher incidence of lymphomas in the Trp53 knockout (KO) mice compared with hemizygous (HEM). In addition, single or complete loss of Trp53 alleles, as well as p53 downregulation, led to a differential increase in basal, LPS- and UVB-induced expression of a plethora of pro-inflammatory cytokine, such as interleukin-12 (Il-12a), TNFα (Tnfa) and interleukin (Il-23a) in bone marrow-derived macrophage cells (BMDMs) compared to WT cells. Interestingly, p53-dependent increased inflammatory gene expression correlated with deregulated expression of the NF-κB pathway inhibitor IκBα. Chromatin immunoprecipitation data revealed decreased p65 binding to Nfkbia in the absence of p53 and p53 binding to Nfkbia promoter, uncovering a novel crosstalk mechanism between p53 and NF-κB transcription factors. Overall, our data suggest that single Trp53 allele loss can drive a sustained inflammatory, DNA damage and oxidative stress response that, over time, facilitate and support carcinogenesis.

Abstract PS19-17: The role of myoglobin in breast cancer

Abstract Breast cancer today is typically seen as malignancy with longer survival due to early-detection diagnostics, yet the cancer remains by far the most common tumor type in adult females. Recently, our group co-discovered myoglobin (MB) to be expressed in luminal cells of healthy and cancerous breast epithelia of human and mice. In human patients, MB positivity emerged as hallmark of the luminal subtype and was directly correlated with estrogen receptor alpha positivity and hence a significantly better prognosis. Cancerous cells’ MB also appears to interact with the known tumor suppressor p53. However, if and how myoglobin itself restricts mammary tumorigenesis is completely unclear. To understand how MB exerts its alleged tumor-suppressive effects and if it impacts response of mammary tumors to chemo- and/or radiotherapy, we examine the molecular role of MB in breast tumor formation and progression through a) in vitro studies (MCF7 breast cancer BrCa cell clones; wildtype (wt) controls versus CRISPR/Cas9-engineered clones of a MB and/or p53 knockout (MBko/p53ko), b) in vivo mouse models to obtain spontaneously forming breast tumors, with or without MB, in a p53 deficient background. Comparing MCF7 BrCa MBko and p53ko clones vs. their corresponding wildtype counterpart supports our hypothesis of a feed forward loop between MB and p53 proteins, besides interfering with estrogen receptor expression. In addition to that, loss of MB was noticed to exert p53-independent upregulation on cell cyclins with more S phase cell population. That was reflected by a significant increase in survival of the MBko cells, in presence or absence of p53. Hypoxic MBko cells exhibited a partial epithelial to mesenchymal transition and hence a more migratory phenotype, relative to MBwt controls. Additionally, MB seems to stimulate apoptosis of the normoxic cancer cells. On the other hand, murine tumors were found to phenocopy human BrCa in the extent of their MB expression. Interestingly, MB-devoid tumors showed a significantly higher proliferation index and fat accumulation. Further studies will look into the clinical significance of that. In conclusion, MB seems to occupy unknown roles in cancer cells beyond or different from its classical O2 storage/transport functions. Unraveling these novel roles in governing tumor development and virulence will hopefully provide innovative strategies for future breast cancer interventions. Citation Format: Mostafa A. Aboouf, Julia Armbruster, Franco Guscetti, Markus Thiersch, Max Gassmann, Thomas A. Gorr. The role of myoglobin in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-17.

The Effect of Neddylation Blockade on Slug-Dependent Cancer Cell Migration Is Regulated by p53 Mutation Status.

Simple SummaryNeddylation is a process in which the small ubiquitin-like molecule NEDD8 is covalently conjugated to target proteins by sequential enzymatic reactions. Because neddylation plays critical roles in regulating cancer growth and migration, it is emerging as an effective therapeutic target. The major tumor suppressor protein p53 reduces cancer cell migration and is inhibited by neddylation. As p53 is lost or mutated in 50% of various cancer types, this study attempted to investigate how neddylation affects cancer cell migration according to p53 status. Neddylation blockade reduced or caused no change in migration of wild type or mutant p53 cancer cell lines. In contrast, neddylation blockade induced migration of p53-null cancer cell lines. These results were mediated by the differential effect of neddylation blockade on the epithelial–mesenchymal transition activator Slug according to p53 status. Thus, the p53 status of cancer cells should be considered when developing neddylation-targeted anticancer drugs.The tumor suppressor protein p53 is frequently inactivated in human malignancies, in which it is associated with cancer aggressiveness and metastasis. Because p53 is heavily involved in epithelial–mesenchymal transition (EMT), a primary step in cell migration, p53 regulation is important for preventing cancer metastasis. p53 function can be modulated by diverse post-translational modifications including neddylation, a reversible process that conjugates NEDD8 to target proteins and inhibits the transcriptional activity of p53. However, the role of p53 in cancer migration by neddylation has not been fully elucidated. In this study, we reported that neddylation blockade induces cell migration depending on p53 status, specifically via the EMT-promoting transcription factor Slug. In cancer cell lines expressing wild type p53, neddylation blockade increased the transcriptional activity of p53 and expression of its downstream genes p21 and MDM2, eventually promoting proteasomal degradation of Slug. In the absence of p53, neddylation blockade increased cell migration by activating the PI3K/Akt/mTOR/Slug signaling axis. Because mutant p53 was transcriptionally inactivated but maintained the ability to bind to Slug, neddylation blockade did not affect the migration of cells expressing mutant p53. Our findings highlight how the p53 expression status influences neddylation-mediated cell migration in multiple cancer cell lines via Slug.

Radiotherapy Delivered before CDK4/6 Inhibitors Mediates Superior Therapeutic Effects in ER+ Breast Cancer.

Recent preclinical data suggest that cyclin-dependent kinase 4/6 (CDK4/6) inhibition may be harnessed to sensitize estrogen receptor-positive (ER+) breast cancer to radiotherapy. However, these findings were obtained in human ER+ breast cancer cell lines exposed to subclinical doses of CDK4/6 inhibitors with limited attention to treatment schedule. We investigated the activity of radiotherapy combined with the prototypic CDK4/6 inhibitor palbociclib placing emphasis on therapeutic schedule. We combined radiotherapy and palbociclib in various doses and therapeutic schedules in human and mouse models of ER+ and ER-negative (ER-) breast cancer, including an immunocompetent mouse model that recapitulates key features of human luminal B breast cancer in women. We assessed proliferation, cell death, cell-cycle control, and clonogenic survival in vitro, as well as tumor growth, overall survival, and metastatic dissemination in vivo. Radiotherapy and palbociclib employed as standalone agents had partial cytostatic effects in vitro, correlating with suboptimal tumor control in vivo. However, while palbociclib delivered before focal radiotherapy provided minimal benefits as compared with either treatment alone, delivering focal radiotherapy before palbociclib mediated superior therapeutic effects, even in the absence of p53. Such superiority manifested in vitro with enhanced cytostasis and loss of clonogenic potential, as well as in vivo with improved local and systemic tumor control. Our preclinical findings demonstrate that radiotherapy delivered before CDK4/6 inhibitors mediates superior antineoplastic effects compared with alternative treatment schedules, calling into question the design of clinical trials administering CDK4/6 inhibitors before radiotherapy in women with ER+ breast cancer.

P53-Independent Induction of p21 Fails to Control Regeneration and Hepatocarcinogenesis in a Murine Liver Injury Model.

Background & AimsA coordinated stress and regenerative response is important after hepatocyte damage. Here, we investigate the phenotypes that result from genetic abrogation of individual components of the checkpoint kinase 2/transformation-related protein 53 (p53)/cyclin-dependent kinase inhibitor 1A (p21) pathway in a murine model of metabolic liver injury.MethodsNitisinone was reduced or withdrawn in Fah-/- mice lacking Chk2, p53, or p21, and survival, tumor development, liver injury, and regeneration were analyzed. Partial hepatectomies were performed and mice were challenged with the Fas antibody Jo2.ResultsIn a model of metabolic liver injury, loss of p53, but not Chk2, impairs the oxidative stress response and aggravates liver damage, indicative of a direct p53-dependent protective effect on hepatocytes. Cell-cycle control during chronic liver injury critically depends on the presence of both p53 and its downstream effector p21. In p53-deficient hepatocytes, unchecked proliferation occurs despite a strong induction of p21, showing a complex interdependency between p21 and p53. The increased regenerative potential in the absence of p53 cannot fully compensate the surplus injury and is not sufficient to promote survival. Despite the distinct phenotypes associated with the loss of individual components of the DNA damage response, gene expression patterns are dominated by the severity of liver injury, but reflect distinct effects of p53 on proliferation and the anti-oxidative stress response.ConclusionsCharacteristic phenotypes result from the genetic abrogation of individual components of the DNA damage-response cascade in a liver injury model. The extent to which loss of gene function can be compensated, or affects injury and proliferation, is related to the level at which the cascade is interrupted. Accession numbers of repository for expression microarray data: GSE156983, GSE156263, GSE156852, and GSE156252.

MiR-26b enhances the sensitivity of hepatocellular carcinoma to Doxorubicin via USP9X-dependent degradation of p53 and regulation of autophagy.

Multi-drug resistance is a major challenge to hepatocellular carcinoma (HCC) treatment, and the over-expression or deletion of microRNA (miRNA) expression is closely related to the drug-resistant properties of various cell lines. However, the underlying molecular mechanisms remain unclear. CCK-8, EdU, flow cytometry, and transmission electron microscopy were performed to determine cell viability, proliferation, apoptosis, autophagic flow, and nanoparticle characterization, respectively. In this study, the results showed that the expression of miR-26b was downregulated following doxorubicin treatment in human HCC tissues. An miR-26b mimic enhanced HCC cell doxorubicin sensitivity, except in the absence of p53 in Hep3B cells. Delivery of the proteasome inhibitor, MG132, reversed the inhibitory effect of miR-26b on the level of p53 following doxorubicin treatment. Tenovin-1 (an MDM2 inhibitor) protected p53 from ubiquitination-mediated degradation only in HepG2 cells with wild type p53. Tenovin-1 pretreatment enhanced HCC cell resistance to doxorubicin when transfected with an miR-26b mimic. Moreover, the miR-26b mimic inhibited doxorubicin-induced autophagy and the autophagy inducer, rapamycin, eliminated the differences in the drug sensitivity effect of miR-26b. In vivo, treatment with sp94dr/miR-26b mimic nanoparticles plus doxorubicin inhibited tumor growth. Our current data indicate that miR-26b enhances HCC cell sensitivity to doxorubicin through diminishing USP9X-mediated p53 de-ubiquitination caused by DNA damaging drugs and autophagy regulation. This miRNA-mediated pathway that modulates HCC will help develop novel therapeutic strategies.

P53 coordinates glucose and choline metabolism during the mesendoderm differentiation of human embryonic stem cells.

Metabolism plays crucial roles in the fate decision of human embryonic stem cells (hESCs). Here, we show that the depletion of p53 in hESCs enhances glycolysis and reduces oxidative phosphorylation, and delays mesendoderm differentiation of hESCs. More intriguingly, the disruption of p53 in hESCs leads to dramatic upregulation of phosphatidylcholine and decrease of total choline in both pluripotent and differentiated state of hESCs, suggesting abnormal choline metabolism in the absence of p53. Collectively, our study reveals the indispensable role of p53 in orchestrating both glucose and lipid metabolism to maintain proper hESC identity.

Deferasirox-Dependent Iron Chelation Enhances Mitochondrial Dysfunction and Restores p53 Signaling by Stabilization of p53 Family Members in Leukemic Cells.

Iron is crucial to satisfy several mitochondrial functions including energy metabolism and oxidative phosphorylation. Patients affected by Myelodysplastic Syndromes (MDS) and acute myeloid leukemia (AML) are frequently characterized by iron overload (IOL), due to continuous red blood cell (RBC) transfusions. This event impacts the overall survival (OS) and it is associated with increased mortality in lower-risk MDS patients. Accordingly, the oral iron chelator Deferasirox (DFX) has been reported to improve the OS and delay leukemic transformation. However, the molecular players and the biological mechanisms laying behind remain currently mostly undefined. The aim of this study has been to investigate the potential anti-leukemic effect of DFX, by functionally and molecularly analyzing its effects in three different leukemia cell lines, harboring or not p53 mutations, and in human primary cells derived from 15 MDS/AML patients. Our findings indicated that DFX can lead to apoptosis, impairment of cell growth only in a context of IOL, and can induce a significant alteration of mitochondria network, with a sharp reduction in mitochondrial activity. Moreover, through a remarkable reduction of Murine Double Minute 2 (MDM2), known to regulate the stability of p53 and p73 proteins, we observed an enhancement of p53 transcriptional activity after DFX. Interestingly, this iron depletion-triggered signaling is enabled by p73, in the absence of p53, or in the presence of a p53 mutant form. In conclusion, we propose a mechanism by which the increased p53 family transcriptional activity and protein stability could explain the potential benefits of iron chelation therapy in terms of improving OS and delaying leukemic transformation.

Cell fate decisions by c-Myc depend on ZBTB5 and p53

The oncoprotein, c-Myc, not only promotes cell proliferation, but can also induce or sensitize cells to apoptosis. However, how c-Myc decides cell fate and which c-Myc downstream target genes are involved remain unknown. Previously, we showed that ZBTB5 (zinc finger and BTB domain-containing 5) is a proto-oncogene that stimulates cell proliferation. ZBTB5 represses p21/CDKN1A by competing with p53 and recruiting corepressor histone deacetylase complexes. Herein, we found that c-Myc directly activates the transcription of ZBTB5. In the absence of p53, ZBTB5 is acetylated at K597 by interacting with p300, and activates transcription of NOXA, which induces apoptosis. In contrast, in the presence of p53, ZBTB5 interacts with p53 and acetylation at ZBTB5 K597 is blocked. ZBTB5 without K597 acetylation interacts with mSin3A/HDAC1 to repress p21/CDKN1A transcription and promote cell proliferation. Cell fate decisions by c-Myc depend on ZBTB5, p53 and p300, and acetylation of ZBTB5 K597.

Abstract PR12: Regulation of the tumor-suppressive miR-29 family by oncogenic MAPK signaling in melanoma

Abstract MicroRNAs are a group of noncoding RNAs that can regulate gene expression by targeting the mRNA of a gene. Many microRNAs respond to different stimuli such as growth signals or cellular stress. The tumor-suppressive miR-29 family (miR-29a, b and c), for example, is induced during aging and senescence as a p53-dependent response to DNA damage. Given the tumor-suppressive potential of miR-29, we sought to examine if its expression is induced by oncogenic stress in a p53-dependent manner. Ectopic overexpression of oncogenic Braf-V600E and Kras-G12D in mouse embryonic fibroblasts (MEFs) using retroviral transduction resulted in increased expression of pri-miR-29a-b1 and, to a lesser extent, pri-miR-29b2~c. While ectopic Kras-G12D overexpression stimulated p53 activity, Braf-V600E failed to elicit a p53 response, suggesting that this oncogene provokes miR-29 expression in a p53-independent manner. Moreover, expression of Braf-V600E at physiologic levels induced miR-29 in the absence of a p53 response. Accordingly, expression of endogenous Braf-V600E stimulated miR-29 in MEFs even when p53 was silenced by RNAi. When Braf-V600E MEFs were treated with a MEK inhibitor, miR-29 levels decreased in both the presence and absence of p53, suggesting that the MAPK pathway downstream of Braf-V600E directly regulates miR-29 transcription. Importantly, TPA-mediated stimulation of the MAPK pathway increased miR-29 levels in human melanocytes. Similarly, expression of Braf-V600E induced miR-29 in human melanocytes and MEK inhibition rescued this effect. These results strongly support the notion that miR-29 transcription is induced by MAPK signaling and suggest that oncogenic BRAF provokes a tumor-suppressive miR-29 checkpoint that needs to be inactivated for further tumor progression. Indeed, human melanoma cells lines express lower levels of miR-29 compared to human melanocytes, and inactivation of miR-29 by a lentiviral sponge in Braf-V600E mutant melanocytes and melanoma cells promotes cell transformation. We are now utilizing our ESC-GEMM platform to generate Braf-V600E; Pten-deficient mice expressing the miR-29 sponge and will assess if counteracting the induction of miR-29 by Braf-V600E promotes melanoma formation in vivo. Our results suggest a biphasic role of miR-29 during melanomagenesis where Braf-V600E-induced miR-29 upregulation prevents the progression of early lesions until miR-29 is downregulated, likely by inactivation of the ARF-p53 axis. This abstract is also being presented as Poster B24. Citation Format: Olga Vera, Ilah Bok, Neel Jasani, Oluwashanu Balogun, Koji Nakamura, Florian Karreth. Regulation of the tumor-suppressive miR-29 family by oncogenic MAPK signaling in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR12.

Abstract 1386: 5-FU-induces PD-L1 in the absence of p53 via ATM dependent activation of STAT3

Abstract Background: 5-Fluorouracil (5-FU) is an antimetabolite DNA damaging chemotherapeutics that forms the backbone for treatment of colorectal cancer (CRC) and currently it is the most commonly used anti-cancer drug. Despite the central role played by p53 in regulating DNA-damage response, its role in regulating response to 5-FU remains unclear. Methods: To better understand how p53 status differentially affects response to 5-FU, we conducted a detailed phenotypic analysis of the effects of 5FU alone or in combination with Oxaliplatin on cell cycle, DNA damage, cell death, DNA repair and consequent signaling across a panel of p53 isogenic, null and mutant CRC cell lines. Results: This revealed that while 5-FU induce both p53 dependent and independent cell death, the effects on cell cycle are profoundly different, with p53 deficient cells accumulating in S-phase with prolongated double strand breaks. This is not observed in p53 proficient cells, which are protected from these effects by induction of the p53 target gene p21/CDKN1A limiting cell cycle and enforcing suppression of cell cycle genes. Notably in both p53 or p21 deficient cells 5-FU results in sustained DNA damage-signaling through ATR and ATM in p53 deficient cells which preferentially arrest in S-phase. Importantly, these cells exhibit significant induction of cell surface expression of programmed death ligand-1 (PD-L1), which is enhanced by ATR inhibition concomitant with increased ATM activation. Further analysis indicates that transcriptional induction of PD-L1 is mediated through STAT3 in an interferon independent manner. Conclusion: This work adds significantly to our understanding of how p53 status impacts 5FU induced cell cycle arrest and response in CRC patients and have important implications for understanding 5-FU response, particularly in future combinations with immune checkpoint inhibitors. Citation Format: Tamas Sessler, Fiammetta Falcone, Peter Gallagher, Timothy Wright, Kienan Savage, Daniel B. Longley, Simon S. McDade. 5-FU-induces PD-L1 in the absence of p53 via ATM dependent activation of STAT3 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1386.

MDM2 as a target for ellagic acid‑mediated suppression of prostate cancer cells invitro.

Prostate cancer (PCa) is the most common cancer in men. Despite the available treatments for PCa, a significant number of patients relapse as the disease becomes hormonal‑independent. p53 is a common tumor suppressor; however, its activity is diminished via the overexpression of murine double minute‑2 (MDM2). The pomegranate, walnuts, and blueberries are widely consumed fruits and nuts that contain several polyphenolic compounds, mainly ellagic acid (EA). The present study focused on the influence of EA on the p53/MDM2 pathway in PCa cell lines. Three human PCa cell lines PCa LNCaP (p53+/+), 22RV1 (p53-/+), and PC3 (p53-/-) harboring different p53 genotypes were used in this research. We found that EA downregulated the gene and protein expression levels of MDM2 and increased the protein expression of p53 as determined by qPCR and western blot analyses. Moreover, by using western blot analysis, we determined that EA increased the protein expression of the p53 target proteins p21, p53 upregulated modulator of apoptosis(PUMA) [also known as Bcl‑2‑binding component3(BBC3)] and Phorbol‑12‑myristate‑13‑acetate‑induced protein 1(NOXA). Furthermore, we found that EA induced apoptosis in the absence of p53 by downregulating MDM2 and X‑linked inhibitor of apoptosis protein (XIAP) protein expressions as determined by western blot analysis. We conclude that EA suppressed PCa cells invitro partly by downregulating MDM2.

Cell cycle arrest and apoptosis are not dependent on p53 prior to p53-dependent embryonic stem cell differentiation.

Previous efforts to determine whether or not the transcription factor and tumor suppressor protein p53 is required for DNA damage-induced apoptosis in pluripotent embryonic stem cells (ESCs) produced contradictory conclusions. To resolve this issue, p53+/+ and p53-/- ESCs derived by two different methods were used to quantify time-dependent changes in nuclear DNA content; annexin-V binding; cell permeabilization; and protein expression, modification, and localization. The results revealed that doxorubicin (Adriamycin [ADR]) concentrations 10 to 40 times less than commonly used in previous studies induced the DNA damage-dependent G2-checkpoint and completed apoptosis within the same time frame, regardless of the presence or absence of p53, p21, and PUMA. Increased ADR concentrations delayed initiation of apoptosis in p53-/- ESCs, but the rates of apoptosis remained equivalent. Similar results were obtained by inducing apoptosis with either staurosporine inhibition of kinase activities or WX8 disruption of lysosome homeostasis. Differentiation of ESCs by LIF deprivation revealed p53-dependent formation of haploid cells, increased genomic stability, and suppression of the G2-checkpoint. Minimal induction of DNA damage now resulted in p53-facilitated apoptosis, but regulation of pluripotent gene expression remained p53-independent. Primary embryonic fibroblasts underwent p53-dependent total cell cycle arrest (a prelude to cell senescence), and p53-independent apoptosis occurred in the presence of 10-fold higher levels of ADR, consistent with previous studies. Taken together, these results reveal that the multiple roles of p53 in cell cycle regulation and apoptosis are first acquired during pluripotent stem cell differentiation.