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1.
Mol Syst Biol ; 20(6): 719-740, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38580884

RESUMO

Tumor suppressor p53 (TP53) is frequently mutated in cancer, often resulting not only in loss of its tumor-suppressive function but also acquisition of dominant-negative and even oncogenic gain-of-function traits. While wild-type p53 levels are tightly regulated, mutants are typically stabilized in tumors, which is crucial for their oncogenic properties. Here, we systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. Most regulators of wild-type p53 also regulate p53 mutants, except for p53 R337H regulators, which are largely private to this mutant. Mechanistically, FBXO42 emerged as a positive regulator for a subset of p53 mutants, working with CCDC6 to control USP28-mediated mutant p53 stabilization. Additionally, C16orf72/HAPSTR1 negatively regulates both wild-type p53 and all tested mutants. C16orf72/HAPSTR1 is commonly amplified in breast cancer, and its overexpression reduces p53 levels in mouse mammary epithelium leading to accelerated breast cancer. This study offers a network perspective on p53 stability regulation, potentially guiding strategies to reinforce wild-type p53 or target mutant p53 in cancer.


Assuntos
Mutação , Estabilidade Proteica , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Humanos , Camundongos , Feminino , Sistemas CRISPR-Cas , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Regulação Neoplásica da Expressão Gênica , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
2.
Cell Rep ; 42(10): 113256, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37847590

RESUMO

It is widely assumed that all normal somatic cells can equally perform homologous recombination (HR) and non-homologous end joining in the DNA damage response (DDR). Here, we show that the DDR in normal mammary gland inherently depends on the epithelial cell lineage identity. Bioinformatics, post-irradiation DNA damage repair kinetics, and clonogenic assays demonstrated luminal lineage exhibiting a more pronounced DDR and HR repair compared to the basal lineage. Consequently, basal progenitors were far more sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis) in both mouse and human mammary epithelium. Furthermore, PARPi sensitivity of murine and human breast cancer cell lines as well as patient-derived xenografts correlated with their molecular resemblance to the mammary progenitor lineages. Thus, mammary epithelial cells are intrinsically divergent in their DNA damage repair capacity and PARPi vulnerability, potentially influencing the clinical utility of this targeted therapy.


Assuntos
Antineoplásicos , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Animais , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antineoplásicos/farmacologia , Reparo do DNA , Recombinação Homóloga , Dano ao DNA
4.
Cancer Discov ; 12(12): 2930-2953, 2022 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-36108220

RESUMO

Systematically investigating the scores of genes mutated in cancer and discerning disease drivers from inconsequential bystanders is a prerequisite for precision medicine but remains challenging. Here, we developed a somatic CRISPR/Cas9 mutagenesis screen to study 215 recurrent "long-tail" breast cancer genes, which revealed epigenetic regulation as a major tumor-suppressive mechanism. We report that components of the BAP1 and COMPASS-like complexes, including KMT2C/D, KDM6A, BAP1, and ASXL1/2 ("EpiDrivers"), cooperate with PIK3CAH1047R to transform mouse and human breast epithelial cells. Mechanistically, we find that activation of PIK3CAH1047R and concomitant EpiDriver loss triggered an alveolar-like lineage conversion of basal mammary epithelial cells and accelerated formation of luminal-like tumors, suggesting a basal origin for luminal tumors. EpiDriver mutations are found in ∼39% of human breast cancers, and ∼50% of ductal carcinoma in situ express casein, suggesting that lineage infidelity and alveogenic mimicry may significantly contribute to early steps of breast cancer etiology. SIGNIFICANCE: Infrequently mutated genes comprise most of the mutational burden in breast tumors but are poorly understood. In vivo CRISPR screening identified functional tumor suppressors that converged on epigenetic regulation. Loss of epigenetic regulators accelerated tumorigenesis and revealed lineage infidelity and aberrant expression of alveogenesis genes as potential early events in tumorigenesis. This article is highlighted in the In This Issue feature, p. 2711.


Assuntos
Neoplasias da Mama , Carcinoma Intraductal não Infiltrante , Humanos , Camundongos , Animais , Feminino , Neoplasias da Mama/patologia , Epigênese Genética , Recidiva Local de Neoplasia/genética , Carcinoma Intraductal não Infiltrante/genética , Transformação Celular Neoplásica/genética
5.
Cancer Cell ; 39(8): 1115-1134.e12, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34270926

RESUMO

Cancer heterogeneity impacts therapeutic response, driving efforts to discover over-arching rules that supersede variability. Here, we define pan-cancer binary classes based on distinct expression of YAP and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we show that opposite pro- or anti-cancer YAP activity functionally defines binary YAPon or YAPoff cancer classes that express or silence YAP, respectively. YAPoff solid cancers are neural/neuroendocrine and frequently RB1-/-, such as retinoblastoma, small cell lung cancer, and neuroendocrine prostate cancer. YAP silencing is intrinsic to the cell of origin, or acquired with lineage switching and drug resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, distinct YAP/TEAD enhancers in YAPoff or YAPon cancers deploy anti-cancer integrin or pro-cancer proliferative programs, respectively. YAP is thus pivotal across cancer, but in opposite ways, with therapeutic implications.


Assuntos
Neoplasias Pulmonares/genética , Carcinoma de Pequenas Células do Pulmão/genética , Fatores de Transcrição de Domínio TEA/genética , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional/genética , Proteínas de Sinalização YAP/genética , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Humanos , Integrinas/metabolismo , Masculino , Camundongos Transgênicos , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Retina/genética , Neoplasias da Retina/patologia , Retinoblastoma/genética , Retinoblastoma/patologia , Proteínas de Ligação a Retinoblastoma/genética , Fatores de Transcrição de Domínio TEA/metabolismo , Ubiquitina-Proteína Ligases/genética , Ensaios Antitumorais Modelo de Xenoenxerto
6.
J Vis Exp ; (165)2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33191932

RESUMO

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to assess therapeutic avenues. However, their time-, labor- and cost-intensive nature limits their utility for systematic analysis of gene function. Recent advances in genome-editing technologies overcome those limitations and allow for the rapid generation of specific gene perturbations directly within specific mouse organs in a multiplexed and rapid manner. Here, we describe a CRISPR/Cas9-based method (Clustered Regularly Interspaced Short Palindromic Repeats) to generate thousands of gene knock-out clones within the epithelium of the skin and oral cavity of mice, and provide a protocol detailing the steps necessary to perform a direct in vivo CRISPR screen for tumor suppressor genes. This approach can be applied to other organs or other CRISPR/Cas9 technologies such as CRISPR-activation or CRISPR-inactivation to study the biological function of genes during tissue homeostasis or in various disease settings.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Testes Genéticos , Boca/metabolismo , Pele/metabolismo , Animais , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Epitélio/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Integrases/metabolismo , Camundongos , Microinjeções , Fenótipo , Ultrassom
7.
Science ; 367(6483): 1264-1269, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32165588

RESUMO

In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent "long tail" mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.


Assuntos
Genes Supressores de Tumor , Predisposição Genética para Doença , Neoplasias de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteínas Supressoras de Tumor/genética , Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Animais , Sistemas CRISPR-Cas , Feminino , Testes Genéticos , Células HEK293 , Humanos , Proteínas com Domínio LIM/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Receptores Notch/genética , Transdução de Sinais/genética
8.
Sci Rep ; 7(1): 17564, 2017 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-29242602

RESUMO

Natriuretic peptides (NPs) play essential roles in the regulation of cardiovascular function. NP effects are mediated by receptors known as NPR-A, NPR-B or NPR-C. NPs have potent effects on regulation of heart rate (HR) by the autonomic nervous system (ANS), but the role of NPR-C in these effects has not been investigated. Accordingly, we have used telemetric ECG recordings in awake, freely moving wildtype and NPR-C knockout (NPR-C-/-) mice and performed heart rate variability (HRV) analysis to assess alterations in sympatho-vagal balance on the heart following loss of NPR-C. Our novel data demonstrate that NPR-C-/- mice are characterized by elevations in HR, reductions in circadian changes in HR and enhanced occurrence of sinus pauses, indicating increased arrhythmogenesis and a loss of HRV. Time domain and frequency domain analyses further demonstrate that HRV is reduced in NPR-C-/- mice in association with a reduction in parasympathetic activity. Importantly, the low frequency to high frequency ratio was increased in NPR-C-/- mice indicating that sympathetic activity is also enhanced. These changes in autonomic regulation were confirmed using atropine and propranolol to antagonize the ANS. These findings illustrate that loss of NPR-C reduces HRV due to perturbations in the regulation of the heart by the ANS.


Assuntos
Sistema Nervoso Autônomo/fisiologia , Frequência Cardíaca , Receptores do Fator Natriurético Atrial/deficiência , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Sistema Nervoso Autônomo/fisiopatologia , Técnicas de Inativação de Genes , Camundongos , Receptores do Fator Natriurético Atrial/genética
10.
Cell Stem Cell ; 21(2): 209-224.e7, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28712938

RESUMO

Glioblastomas exhibit a hierarchical cellular organization, suggesting that they are driven by neoplastic stem cells that retain partial yet abnormal differentiation potential. Here, we show that a large subset of patient-derived glioblastoma stem cells (GSCs) express high levels of Achaete-scute homolog 1 (ASCL1), a proneural transcription factor involved in normal neurogenesis. ASCL1hi GSCs exhibit a latent capacity for terminal neuronal differentiation in response to inhibition of Notch signaling, whereas ASCL1lo GSCs do not. Increasing ASCL1 levels in ASCL1lo GSCs restores neuronal lineage potential, promotes terminal differentiation, and attenuates tumorigenicity. ASCL1 mediates these effects by functioning as a pioneer factor at closed chromatin, opening new sites to activate a neurogenic gene expression program. Directing GSCs toward terminal differentiation may provide therapeutic applications for a subset of GBM patients and strongly supports efforts to restore differentiation potential in GBM and other cancers.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias Encefálicas/patologia , Carcinogênese/patologia , Linhagem da Célula , Cromatina/metabolismo , Glioblastoma/patologia , Neurônios/patologia , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Neoplasias Encefálicas/genética , Carcinogênese/genética , Diferenciação Celular/genética , Progressão da Doença , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neurônios/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Análise de Sequência de RNA , Regulação para Cima/genética
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