RESUMO
In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.
Assuntos
Dano ao DNA , RNA Polimerase II/metabolismo , Reparo do DNA , Células HEK293 , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Ubiquitinação , Raios UltravioletaRESUMO
Computational analysis of bio-images by deep learning (DL) algorithms has made exceptional progress in recent years and has become much more accessible to non-specialists with the development of ready-to-use tools. The study of oogenesis mechanisms and female reproductive success has also recently benefited from the development of efficient protocols for three-dimensional (3D) imaging of ovaries. Such datasets have a great potential for generating new quantitative data but are, however, complex to analyze due to the lack of efficient workflows for 3D image analysis. Here, we have integrated two existing open-source DL tools, Noise2Void and Cellpose, into an analysis pipeline dedicated to 3D follicular content analysis, which is available on Fiji. Our pipeline was developed on larvae and adult medaka ovaries but was also successfully applied to different types of ovaries (trout, zebrafish and mouse). Image enhancement, Cellpose segmentation and post-processing of labels enabled automatic and accurate quantification of these 3D images, which exhibited irregular fluorescent staining, low autofluorescence signal or heterogeneous follicles sizes. In the future, this pipeline will be useful for extensive cellular phenotyping in fish or mammals for developmental or toxicology studies.
Assuntos
Aprendizado Profundo , Feminino , Animais , Camundongos , Ovário/diagnóstico por imagem , Peixe-Zebra , Imageamento Tridimensional/métodos , Processamento de Imagem Assistida por Computador/métodos , MamíferosRESUMO
BACKGROUND: In vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies. METHODS: To facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes. RESULTS: We demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination. CONCLUSIONS: Our study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.
Assuntos
Copépodes , Neoplasias Pulmonares , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Copépodes/genética , Copépodes/metabolismo , Edição de Genes , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Neoplasias Pulmonares/genética , CamundongosRESUMO
Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Different approaches have been described to study RNAPII poly-ubiquitylation and degradation, each method with its own advantages and caveats. Here, we describe optimised strategies for detecting ubiquitylated RNAPII and studying its degradation, but these protocols are suitable for studying other ubiquitylated proteins as well.
Assuntos
RNA Polimerase II/análise , RNA Polimerase II/metabolismo , Ubiquitinação , Animais , Dano ao DNA , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/genética , Transcrição Gênica , Raios Ultravioleta , Leveduras/enzimologia , Leveduras/genética , Leveduras/metabolismoRESUMO
Tumor progression to a metastatic and ultimately lethal stage relies on a tumor-supporting microenvironment that is generated by reciprocal communication between tumor and stromal host cells. The tumor-stroma crosstalk is instructed by the genetic alterations of the tumor cells-the most frequent being mutations in the gene Tumor protein p53 (TP53) that are clinically correlated with metastasis, drug resistance and poor patient survival. The crucial mediators of tumor-stroma communication are tumor-derived extracellular vesicles (EVs), in particular exosomes, which operate both locally within the primary tumor and in distant organs, at pre-metastatic niches as the future sites of metastasis. Here, we review how wild-type and mutant p53 proteins control the secretion, size, and especially the RNA and protein cargo of tumor-derived EVs. We highlight how EVs extend the cell-autonomous tumor suppressive activity of wild-type p53 into the tumor microenvironment (TME), and how mutant p53 proteins switch EVs into oncogenic messengers that reprogram tumor-host communication within the entire organism so as to promote metastatic tumor cell dissemination.
Assuntos
Comunicação Celular , Vesículas Extracelulares/metabolismo , Neoplasias/patologia , Microambiente Tumoral , Proteína Supressora de Tumor p53/metabolismo , Animais , Humanos , Metástase Neoplásica , Neoplasias/metabolismoRESUMO
The transcription factor FOXL2 is required in ovarian somatic cells for female fertility. Differential timing of Foxl2 deletion, in embryonic versus adult mouse ovary, leads to distinctive outcomes, suggesting different roles across development. Here, we comprehensively investigated FOXL2's role through a multi-omics approach to characterize gene expression dynamics and chromatin accessibility changes, coupled with genome-wide identification of FOXL2 targets and on-chromatin interacting partners in somatic cells across ovarian development. We found that FOXL2 regulates more targets postnatally, through interaction with factors regulating primordial follicle formation and steroidogenesis. Deletion of one interactor, ubiquitin-specific protease 7 (Usp7), results in impairment of somatic cell differentiation, germ cell nest breakdown, and ovarian development, leading to sterility. Our datasets constitute a comprehensive resource for exploration of the molecular mechanisms of ovarian development and causes of female infertility.
Assuntos
Fatores de Transcrição Forkhead , Ovário , Animais , Camundongos , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Ovário/metabolismo , Folículo Ovariano/metabolismo , Regulação da Expressão Gênica , Cromatina/genética , Cromatina/metabolismoRESUMO
BACKGROUND: TP53, encoding the tumor suppressor p53, is frequently mutated in various cancers, producing mutant p53 proteins (mutp53) which can exhibit neomorphic, gain-of-function properties. The latter transform p53 into an oncoprotein that promotes metastatic tumor progression via downstream effectors such as ENTPD5, an endoplasmic reticulum UDPase involved in the calnexin/calreticulin cycle of N-glycoprotein biosynthesis. Elucidating the mechanisms underlying the pro-metastatic functions of the mutp53-ENTPD5 axis is crucial for developing targeted therapies for aggressive metastatic cancer. METHODS: We analyzed pancreatic, lung, and breast adenocarcinoma cells with p53 missense mutations to study the impact of mutp53 and ENTPD5 on the N-glycoproteins integrin-α5 (ITGA5) and integrin-ß1 (ITGB1), which heterodimerize to form the key fibronectin receptor. We assessed the role of the mutp53-ENTPD5 axis in integrin-dependent tumor-stroma interactions and tumor cell motility using adhesion, migration, and invasion assays, identifying and validating therapeutic intervention targets. We employed an orthotopic xenograft model of pancreatic ductal adenocarcinoma to examine in vivo targeting of mutp53-ENTPD5-mediated ITGA5 regulation for cancer therapy. RESULTS: Mutp53 depletion diminished ITGA5 and ITGB1 expression and impaired tumor cell adhesion, migration, and invasion, rescued by ENTPD5. The mutp53-ENTPD5 axis maintained ITGA5 expression and function via the calnexin/calreticulin cycle. Targeting this axis using ITGA5-blocking antibodies, α-glucosidase inhibitors, or pharmacological degradation of mutp53 by HSP90 inhibitors, such as Ganetespib, effectively inhibited ITGA5-mediated cancer cell motility in vitro. In the orthotopic xenograft model, Ganetespib reduced ITGA5 expression and metastasis in an ENTPD5-dependent manner. CONCLUSIONS: The mutp53-ENTPD5 axis fosters ITGA5 and ITGB1 expression and tumor cell motility through the calnexin/calreticulin cycle, contributing to cancer metastasis. ITGA5-blocking antibodies or α-glucosidase inhibitors target this axis and represent potential therapeutic options worth exploring in preclinical models. The pharmacologic degradation of mutp53 by HSP90 inhibitors effectively blocks ENTPD5-ITGA5-mediated cancer cell motility and metastasis in vivo, warranting further clinical evaluation in p53-mutant cancers. This research underscores the significance of understanding the complex interplay between mutp53, ENTPD5, and the calnexin/calreticulin cycle in integrin-mediated metastatic tumor progression, offering valuable insights for the development of potential therapeutic strategies.
Assuntos
Adenocarcinoma , Antineoplásicos , Animais , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Calnexina/genética , Calnexina/metabolismo , Integrina alfa5/metabolismo , Calreticulina/metabolismo , Anticorpos Bloqueadores/metabolismo , Inibidores de Glicosídeo Hidrolases , Linhagem Celular Tumoral , Chaperonas Moleculares/metabolismo , Modelos Animais de Doenças , Pirofosfatases/metabolismo , Proteínas Oncogênicas/metabolismoRESUMO
The tumor suppressive transcription factor p53 is frequently inactivated in cancer cells by missense mutations that cluster in the DNA binding domain. 30% hit mutational hotspot residues, resulting in a complete loss of transcriptional activity and mutant p53-driven chemotherapy resistance. Of the remaining 70% of non-hotspot mutants, many are partial loss-of-function (partial-LOF) mutants with residual transcriptional activity. The therapeutic consequences of a partial-LOF have remained largely elusive. Using a p53 mutation engineered to reduce DNA binding, we demonstrate that partial-LOF is sufficient to enhance oncogene-driven tumorigenesis in mouse models of lung and pancreatic ductal adenocarcinoma and acute myeloid leukemia. Interestingly, mouse and human tumors with partial-LOF mutations showed mutant p53 protein accumulation similar as known for hotspot mutants. Different from the chemotherapy resistance caused by p53-loss, the partial-LOF mutant sensitized to an apoptotic chemotherapy response and led to a survival benefit. Mechanistically, the pro-apoptotic transcriptional activity of mouse and human partial-LOF mutants was rescued at high mutant protein levels, suggesting that accumulation of partial-LOF mutants enables the observed apoptotic chemotherapy response. p53 non-hotspot mutants with partial-LOF, therefore, represent tumorigenic p53 mutations that need to be distinguished from other mutations because of their beneficial impact on survival in a therapy context.
Assuntos
Proteína Supressora de Tumor p53RESUMO
BACKGROUND: Impaired p53 function is one of the central molecular features of a tumor cell and even a partial reduction in p53 activity can increase the cancer risk in mice and men. From a therapeutic perspective it is noteworthy that tumor cells often become addicted to the absence of p53 providing a rationale for developing p53 reactivating compounds to treat cancer patients. Unfortunately, many of the compounds that are currently undergoing preclinical and clinical testing fail to fully reactivate mutant p53 proteins, raising the crucial question: how much p53 activity is needed to elicit a therapeutic effect? METHODS: We have genetically modelled partial p53 reactivation using knock-in mice with inducible expression of the p53 variant E177R. This variant has a reduced ability to bind and transactivate target genes and consequently causes moderate cancer susceptibility. We have generated different syngeneically transplanted and autochthonous mouse models of p53-deficient acute myeloid leukemia and B or T cell lymphoma. After cancer manifestation we have activated E177R expression and analyzed the in vivo therapy response by bioluminescence or magnetic resonance imaging. The molecular response was further characterized in vitro by assays for gene expression, proliferation, senescence, differentiation, apoptosis and clonogenic growth. RESULTS: We report the conceptually intriguing observation that the p53 variant E177R, which promotes de novo leukemia and lymphoma formation, inhibits proliferation and viability, induces immune cell infiltration and triggers cancer regression in vivo when introduced into p53-deficient leukemia and lymphomas. p53-deficient cancer cells proved to be so addicted to the absence of p53 that even the low-level activity of E177R is detrimental to cancer growth. CONCLUSIONS: The observation that a partial loss-of-function p53 variant promotes tumorigenesis in one setting and induces regression in another, underlines the highly context-specific effects of individual p53 mutants. It further highlights the exquisite sensitivity of cancer cells to even small changes in p53 activity and reveals that changes in activity level are more important than the absolute level. As such, the study encourages ongoing research efforts into mutant p53 reactivating drugs by providing genetic proof-of-principle evidence that incomplete p53 reactivation may suffice to elicit a therapeutic response.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Apoptose , Carcinogênese , Humanos , Proteínas Mutantes , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The development of sexually dimorphic gonads is a unique process that starts with the specification of the bipotential genital ridges and culminates with the development of fully differentiated ovaries and testes in females and males, respectively. Research on sex determination has been mostly focused on the identification of sex determination genes, the majority of which encode for proteins and specifically transcription factors such as SOX9 in the testes and FOXL2 in the ovaries. Our understanding of which factors may be critical for sex determination have benefited from the study of human disorders of sex development (DSD) and animal models, such as the mouse and the goat, as these often replicate the same phenotypes observed in humans when mutations or chromosomic rearrangements arise in protein-coding genes. Despite the advances made so far in explaining the role of key factors such as SRY, SOX9, and FOXL2 and the genes they control, what may regulate these factors upstream is not entirely understood, often resulting in the inability to correctly diagnose DSD patients. The role of non-coding DNA, which represents 98% of the human genome, in sex determination has only recently begun to be fully appreciated. In this review, we summarize the current knowledge on the long-range regulation of 2 important sex determination genes, SOX9 and FOXL2, and discuss the challenges that lie ahead and the many avenues of research yet to be explored in the sex determination field.