BACKGROUND: Synonymous mutations (SMs) change the mRNA nucleotide sequences without altering the corresponding amino acid sequence and are usually overlooked due to their perceived lack of influence on protein function. However, emerging reports suggest that SMs play a significant role in disease development and progression. METHODS: Whole exome sequencing, RNA-sequencing, and droplet digital PCR were performed to identify the SMs from the malignant glioma patients. MutaRNA was used to predict the effect of SMs on RNA structure in silico. SHAPE-MaP was performed to probe and assess the effect of SMs on RNA structure in-cellulo. RESULTS: Here, we report that a Cancer-Associated SM in TP53 codon valine 203 (CASM203) results in the induction of the alternative translation initiated p53 protein isoform, p47. In-cell high-throughput RNA structural mapping showed that CASM203 mimics the Protein Kinase RNA-Like ER Kinase (PERK)-mediated p53 mRNA secondary structure that induces p47 expression of during the unfolded protein response (UPR). CONCLUSIONS: Overall, the single gain-of-function SM mimics the UPR-mediated p53 stress response, by generating RNA secondary structures akin to the PERK-mediated p53 mRNA structural switch. This illustrates the link between RNA structures and cellular biology and underscores the importance of SMs in cancer biology and their potential to further refine genetic diagnostics.

• Publication type
• Journal Article MeSH

BACKGROUND: The ATM kinase constitutes a master regulatory hub of DNA damage and activates the p53 response pathway by phosphorylating the MDM2 protein, which develops an affinity for the p53 mRNA secondary structure. Disruption of this interaction prevents the activation of the nascent p53. The link of the MDM2 protein-p53 mRNA interaction with the upstream DNA damage sensor ATM kinase and the role of the p53 mRNA in the DNA damage sensing mechanism, are still highly anticipated. METHODS: The proximity ligation assay (PLA) has been extensively used to reveal the sub-cellular localisation of the protein-mRNA and protein-protein interactions. ELISA and co-immunoprecipitation confirmed the interactions in vitro and in cells. RESULTS: This study provides a novel mechanism whereby the p53 mRNA interacts with the ATM kinase enzyme and shows that the L22L synonymous mutant, known to alter the secondary structure of the p53 mRNA, prevents the interaction. The relevant mechanistic roles in the DNA Damage Sensing pathway, which is linked to downstream DNA damage response, are explored. Following DNA damage (double-stranded DNA breaks activating ATM), activated MDMX protein competes the ATM-p53 mRNA interaction and prevents the association of the p53 mRNA with NBS1 (MRN complex). These data also reveal the binding domains and the phosphorylation events on ATM that regulate the interaction and the trafficking of the complex to the cytoplasm. CONCLUSION: The presented model shows a novel interaction of ATM with the p53 mRNA and describes the link between DNA Damage Sensing with the downstream p53 activation pathways; supporting the rising functional implications of synonymous mutations altering secondary mRNA structures.

• Keywords
• DNA Damage Sensing, Genotoxic stress, MDM2, MRN complex, Precision medicine, RNA secondary structure, Synonymous mutations,
• MeSH
• Ataxia Telangiectasia Mutated Proteins MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 MeSH
• DNA Repair MeSH
• Polynucleotide 5'-Hydroxyl-Kinase * MeSH
• DNA Damage MeSH
• Proto-Oncogene Proteins c-mdm2 * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATM protein, human MeSH Browser
• Ataxia Telangiectasia Mutated Proteins MeSH
• Tumor Suppressor Protein p53 MeSH
• Polynucleotide 5'-Hydroxyl-Kinase * MeSH
• Proto-Oncogene Proteins c-mdm2 * MeSH

Cellular stress conditions activate p53-dependent pathways to counteract the inflicted damage. To achieve the required functional diversity, p53 is subjected to numerous post-translational modifications and the expression of isoforms. Little is yet known how p53 has evolved to respond to different stress pathways. The p53 isoform p53/47 (p47 or ΔNp53) is linked to aging and neural degeneration and is expressed in human cells via an alternative cap-independent translation initiation from the 2nd in-frame AUG at codon 40 (+118) during endoplasmic reticulum (ER) stress. Despite an AUG codon in the same location, the mouse p53 mRNA does not express the corresponding isoform in either human or mouse-derived cells. High-throughput in-cell RNA structure probing shows that p47 expression is attributed to PERK kinase-dependent structural alterations in the human p53 mRNA, independently of eIF2α. These structural changes do not take place in murine p53 mRNA. Surprisingly, PERK response elements required for the p47 expression are located downstream of the 2nd AUG. The data show that the human p53 mRNA has evolved to respond to PERK-mediated regulation of mRNA structures in order to control p47 expression. The findings highlight how p53 mRNA co-evolved with the function of the encoded protein to specify p53-activities under different cellular conditions.

• MeSH
• eIF-2 Kinase genetics   metabolism   MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mice MeSH
• Tumor Suppressor Protein p53 * genetics   metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Protein Isoforms metabolism   MeSH
• Endoplasmic Reticulum Stress * genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• eIF-2 Kinase MeSH
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 * MeSH
• Protein Isoforms MeSH

The p53 tumor suppressor is a transcription factor with roles in cell development, apoptosis, oncogenesis, aging, and homeostasis in response to stresses and infections. p53 is tightly regulated by the MDM2 E3 ubiquitin ligase. The p53-MDM2 pathway has coevolved, with MDM2 remaining largely conserved, whereas the TP53 gene morphed into various isoforms. Studies on prevertebrate ancestral homologs revealed the transition from an environmentally induced mechanism activating p53 to a tightly regulated system involving cell signaling. The evolution of this mechanism depends on structural changes in the interacting protein motifs. Elephants such as Loxodonta africana constitute ideal models to investigate this coevolution as they are large and long-living as well as having 20 copies of TP53 isoformic sequences expressing a variety of BOX-I MDM2-binding motifs. Collectively, these isoforms would enhance sensitivity to cellular stresses, such as DNA damage, presumably accounting for strong cancer defenses and other adaptations favoring healthy aging. Here we investigate the molecular evolution of the p53-MDM2 system by combining in silico modeling and in vitro assays to explore structural and functional aspects of p53 isoforms retaining the MDM2 interaction, whereas forming distinct pools of cell signaling. The methodology used demonstrates, for the first time that in silico docking simulations can be used to explore functional aspects of elephant p53 isoforms. Our observations elucidate structural and mechanistic aspects of p53 regulation, facilitate understanding of complex cell signaling, and suggest testable hypotheses of p53 evolution referencing Peto's Paradox.

• Keywords
• Loxodonta africana, Peto’s Paradox, intrinsic specificity, lifespan, model, molecular evolution, p53 retrogenes, structural variations,
• MeSH
• Genes, p53 MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Neoplasms * genetics   MeSH
• Protein Isoforms genetics   metabolism   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   metabolism   MeSH
• Elephants * genetics   metabolism   MeSH
• Ubiquitination MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor Protein p53 MeSH
• Protein Isoforms MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH

Cancer is the second leading cause of death globally. One of the main hallmarks in cancer is the functional deregulation of crucial molecular pathways via driver genetic events that lead to abnormal gene expression, giving cells a selective growth advantage. Driver events are defined as mutations, fusions and copy number alterations that are causally implicated in oncogenesis. Molecular analysis on tissues that have originated from a wide range of anatomical areas has shown that mutations in different members of several pathways are implicated in different cancer types. In recent decades, significant efforts have been made to incorporate this knowledge into daily medical practice, providing substantial insight towards clinical diagnosis and personalized therapies. However, since there is still a strong need for more effective drug development, a deep understanding of the involved signaling mechanisms and the interconnections between these pathways is highly anticipated. Here, we perform a systemic analysis on cancer patients included in the Pan-Cancer Atlas project, with the aim to select the ten most highly mutated signaling pathways (p53, RTK-RAS, lipids metabolism, PI-3-Kinase/Akt, ubiquitination, b-catenin/Wnt, Notch, cell cycle, homology directed repair (HDR) and splicing) and to provide a detailed description of each pathway, along with the corresponding therapeutic applications currently being developed or applied. The ultimate scope is to review the current knowledge on highly mutated pathways and to address the attractive perspectives arising from ongoing experimental studies for the clinical implementation of personalized medicine.

• Keywords
• NGS, cancer patients, clinical implementation, molecular oncology, mutations, precision medicine, tumor,
• Publication type
• Journal Article MeSH
• Review MeSH

Since the discovery of the first MDM2 inhibitors, we have gained deeper insights into the cellular roles of MDM2 and p53. In this review, we focus on MDM2 inhibitors that bind to the p53-binding domain of MDM2 and aim to disrupt the binding of MDM2 to p53. We describe the basic mechanism of action of these MDM2 inhibitors, such as nutlin-3a, summarise the determinants of sensitivity to MDM2 inhibition from p53-dependent and p53-independent points of view and discuss the problems with innate and acquired resistance to MDM2 inhibition. Despite progress in MDM2 inhibitor design and ongoing clinical trials, their broad use in cancer treatment is not fulfilling expectations in heterogenous human cancers. We assess the MDM2 inhibitor types in clinical trials and provide an overview of possible sources of resistance to MDM2 inhibition, underlining the need for patient stratification based on these aspects to gain better clinical responses, including the use of combination therapies for personalised medicine.

• Keywords
• Combination therapy, MDM2, MDM2 inhibitor, Nutlin-3a, Personalised medicine, Resistance, p53,
• MeSH
• Drug Resistance, Bacterial drug effects   physiology   MeSH
• Molecular Targeted Therapy methods   MeSH
• Clinical Trials as Topic MeSH
• Humans MeSH
• Tumor Suppressor Protein p53 antagonists & inhibitors   genetics   metabolism   MeSH
• Neoplasms drug therapy   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Proto-Oncogene Proteins c-mdm2 antagonists & inhibitors   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• MDM2 protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Antineoplastic Agents MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• TP53 protein, human MeSH Browser

Human cells are subjected to continuous challenges by different genotoxic stress attacks. DNA damage leads to erroneous mutations, which can alter the function of oncogenes or tumor suppressors, resulting in cancer development. To circumvent this, cells activate the DNA damage response (DDR), which mainly involves cell cycle regulation and DNA repair processes. The tumor suppressor p53 plays a pivotal role in the DDR by halting the cell cycle and facilitating the DNA repair processes. Various pathways and factors participating in the detection and repair of DNA have been described, including scores of RNA-binding proteins (RBPs) and RNAs. It has become increasingly clear that p53's role is multitasking, and p53 mRNA regulation plays a prominent part in the DDR. This review is aimed at covering the p53 RNA metabolism linked to the DDR and highlights the recent findings.

• Keywords
• ATM kinase, DNA damage response, MDM2, RNA metabolism, RNA-binding proteins, mRNA translation, p53,
• MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Mutation MeSH
• Tumor Suppressor Protein p53 genetics   metabolism   MeSH
• Untranslated Regions MeSH
• DNA Repair * physiology   MeSH
• DNA Damage * MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 MeSH
• Untranslated Regions MeSH
• RNA-Binding Proteins MeSH

The p53 and Mouse double minute 2 (MDM2) proteins are hubs in extensive networks of interactions with multiple partners and functions. Intrinsically disordered regions help to adopt function-specific structural conformations in response to ligand binding and post-translational modifications. Different techniques have been used to dissect interactions of the p53-MDM2 pathway, in vitro, in vivo, and in situ each having its own advantages and disadvantages. This review uses the p53-MDM2 to show how different techniques can be employed, illustrating how a combination of in vitro and in vivo techniques is highly recommended to study the spatio-temporal location and dynamics of interactions, and to address their regulation mechanisms and functions. By using well-established techniques in combination with more recent advances, it is possible to rapidly decipher complex mechanisms, such as the p53 regulatory pathway, and to demonstrate how protein and nucleotide ligands in combination with post-translational modifications, result in inter-allosteric and intra-allosteric interactions that govern the activity of the protein complexes and their specific roles in oncogenesis. This promotes elegant therapeutic strategies that exploit protein dynamics to target specific interactions.

• Keywords
• ATM *, DNA damage response *, MDM2 *, MDMX *, p53 *, p53 mRNA *, post-translational modification *, protein-RNA interactions *, protein-protein interactions *,
• MeSH
• Phosphorylation genetics   MeSH
• Nuclear Proteins MeSH
• Humans MeSH
• Mice MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• DNA Damage genetics   MeSH
• Protein Processing, Post-Translational genetics   MeSH
• Cell Cycle Proteins genetics   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   MeSH
• Protein Binding genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Nuclear Proteins MeSH
• MDM2 protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Cell Cycle Proteins MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH

Cell growth requires a high level of protein synthesis and oncogenic pathways stimulate cell proliferation and ribosome biogenesis. Less is known about how cells respond to dysfunctional mRNA translation and how this feeds back into growth regulatory pathways. The Epstein-Barr virus (EBV)-encoded EBNA1 causes mRNA translation stress in cis that activates PI3Kδ. This leads to the stabilization of MDM2, induces MDM2's binding to the E2F1 mRNA and promotes E2F1 translation. The MDM2 serine 166 regulates the interaction with the E2F1 mRNA and deletion of MDM2 C-terminal RING domain results in a constitutive E2F1 mRNA binding. Phosphorylation on serine 395 following DNA damage instead regulates p53 mRNA binding to its RING domain and prevents the E2F1 mRNA interaction. The p14Arf tumour suppressor binds MDM2 and in addition to preventing degradation of the p53 protein it also prevents the E2F1 mRNA interaction. The data illustrate how two MDM2 domains selectively bind specific mRNAs in response to cellular conditions to promote, or suppress, cell growth and how p14Arf coordinates MDM2's activity towards p53 and E2F1. The data also show how EBV via EBNA1-induced mRNA translation stress targets the E2F1 and the MDM2 - p53 pathway.

• MeSH
• Cell Cycle genetics   MeSH
• Phosphorylation genetics   MeSH
• Carcinogenesis genetics   MeSH
• Humans MeSH
• RNA, Messenger genetics   MeSH
• Tumor Suppressor Protein p14ARF genetics   MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Neoplasms genetics   virology   MeSH
• Oncogenes genetics   MeSH
• DNA Damage genetics   MeSH
• Cell Proliferation genetics   MeSH
• Protein Domains genetics   MeSH
• Proto-Oncogene Proteins c-mdm2 genetics   MeSH
• RNA Recognition Motif Proteins genetics   MeSH
• E2F1 Transcription Factor genetics   MeSH
• Genes, Tumor Suppressor MeSH
• Herpesvirus 4, Human genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• E2F1 protein, human MeSH Browser
• MDM2 protein, human MeSH Browser
• RNA, Messenger MeSH
• Tumor Suppressor Protein p14ARF MeSH
• Tumor Suppressor Protein p53 MeSH
• Proto-Oncogene Proteins c-mdm2 MeSH
• RNA Recognition Motif Proteins MeSH
• TP53 protein, human MeSH Browser
• E2F1 Transcription Factor MeSH

The tumor suppressor protein p53 orchestrates cellular responses to a vast number of stresses, with DNA damage and oncogenic activation being some of the best described. The capacity of p53 to control cellular events such as cell cycle progression, DNA repair, and apoptosis, to mention some, has been mostly linked to its role as a transcription factor. However, how p53 integrates different signaling cascades to promote a particular pathway remains an open question. One way to broaden its capacity to respond to different stimuli is by the expression of isoforms that can modulate the activities of the full-length protein. One of these isoforms is p47 (p53/47, Δ40p53, p53ΔN40), an alternative translation initiation variant whose expression is specifically induced by the PERK kinase during the Unfolded Protein Response (UPR) following Endoplasmic Reticulum stress. Despite the increasing knowledge on the p53 pathway, its activity when the translation machinery is globally suppressed during the UPR remains poorly understood. Here, we focus on the expression of p47 and we propose that the alternative initiation of p53 mRNA translation offers a unique condition-dependent mechanism to differentiate p53 activity to control cell homeostasis during the UPR. We also discuss how the manipulation of these processes may influence cancer cell physiology in light of therapeutic approaches.

• Keywords
• ER stress, UPR, mRNA translation, p47, p53,
• Publication type
• Journal Article MeSH
• Review MeSH