RESUMO
Lung cancer is the leading cause of cancer-related death worldwide. Lung adenocarcinoma (LUAD), the most common histological subtype, accounts for 40% of all cases. While existing genetically engineered mouse models (GEMMs) recapitulate the histological progression and transcriptional evolution of human LUAD, they are time-consuming and technically demanding. In contrast, cell line transplant models are fast and flexible, but these models fail to capture the full spectrum of disease progression. Organoid technologies provide a means to create next-generation cancer models that integrate the most advantageous features of autochthonous and transplant-based systems. However, robust and faithful LUAD organoid platforms are currently lacking. Here, we describe optimized conditions to continuously expand murine alveolar type 2 (AT2) cells, a prominent cell of origin for LUAD, in organoid culture. These organoids display canonical features of AT2 cells, including marker gene expression, the presence of lamellar bodies, and an ability to differentiate into the AT1 lineage. We used this system to develop flexible and versatile immunocompetent organoid-based models of KRAS, BRAF, and ALK mutant LUAD. Notably, organoid-based tumors display extensive burden and complete penetrance and are histopathologically indistinguishable from their autochthonous counterparts. Altogether, this organoid platform is a powerful, versatile new model system to study LUAD.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos , Organoides , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores Proteína Tirosina Quinases/metabolismoRESUMO
The state and behaviour of a cell can be influenced by both genetic and environmental factors. In particular, tumour progression is determined by underlying genetic aberrations1-4 as well as the makeup of the tumour microenvironment5,6. Quantifying the contributions of these factors requires new technologies that can accurately measure the spatial location of genomic sequence together with phenotypic readouts. Here we developed slide-DNA-seq, a method for capturing spatially resolved DNA sequences from intact tissue sections. We demonstrate that this method accurately preserves local tumour architecture and enables the de novo discovery of distinct tumour clones and their copy number alterations. We then apply slide-DNA-seq to a mouse model of metastasis and a primary human cancer, revealing that clonal populations are confined to distinct spatial regions. Moreover, through integration with spatial transcriptomics, we uncover distinct sets of genes that are associated with clone-specific genetic aberrations, the local tumour microenvironment, or both. Together, this multi-modal spatial genomics approach provides a versatile platform for quantifying how cell-intrinsic and cell-extrinsic factors contribute to gene expression, protein abundance and other cellular phenotypes.
Assuntos
Células Clonais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Genômica/métodos , Animais , Células Clonais/patologia , Variações do Número de Cópias de DNA/genética , Humanos , Camundongos , Fenótipo , RNA-Seq , Análise de Sequência de DNA , Transcrição Gênica , TranscriptomaRESUMO
Parkinson's disease (PD) is characterized by the accumulation of misfolded alpha-synuclein (α-syn) protein, forming intraneuronal Lewy body (LB) inclusions. The α-syn preformed fibril (PFF) model of PD recapitulates α-syn aggregation, progressive nigrostriatal degeneration and motor dysfunction; however, little is known about the time course of PFF-induced alterations in basal and evoked dopamine (DA). In vivo microdialysis is well suited for identifying small changes in neurotransmitter levels over extended periods. In the present study, adult male Fischer 344 rats received unilateral, intrastriatal injections of either α-syn PFFs or phosphate-buffered saline (PBS). At 4 or 8 months post-injection (p.i.), animals underwent in vivo microdialysis to evaluate basal extracellular striatal DA and metabolite levels, local KCl-evoked striatal DA release and the effects of systemic levodopa (l-DOPA). Post-mortem analysis demonstrated equivalent PFF-induced reductions in tyrosine hydroxylase (TH) immunoreactive nigral neurons (~50%) and striatal TH (~20%) at both time points. Compared with reduction in striatal TH, reduction in striatal dopamine transporter (DAT) was more pronounced and progressed between the 4- and 8-month p.i. intervals (36% â 46%). Significant PFF-induced deficits in basal and evoked striatal DA, as well as deficits in motor performance, were not observed until 8 months p.i. Responses to l-DOPA did not differ regardless of PBS or PFF treatment. These results suggest that basal and evoked striatal DA are maintained for several months following PFF injection, with loss of both associated with motor dysfunction. Our studies provide insight into the time course and magnitude of PFF-induced extracellular dopaminergic deficits in the striatum.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Ratos , Masculino , Animais , alfa-Sinucleína/metabolismo , Dopamina/metabolismo , Levodopa/farmacologia , Microdiálise , Substância Negra/metabolismo , Doença de Parkinson/metabolismoRESUMO
Physiologic activation of estrogen receptor α (ERα) is mediated by estradiol (E2) binding in the ligand-binding pocket of the receptor, repositioning helix 12 (H12) to facilitate binding of coactivator proteins in the unoccupied coactivator binding groove. In breast cancer, activation of ERα is often observed through point mutations that lead to the same H12 repositioning in the absence of E2. Through expanded genetic sequencing of breast cancer patients, we identified a collection of mutations located far from H12 but nonetheless capable of promoting E2-independent transcription and breast cancer cell growth. Using machine learning and computational structure analyses, this set of mutants was inferred to act distinctly from the H12-repositioning mutants and instead was associated with conformational changes across the ERα dimer interface. Through both in vitro and in-cell assays of full-length ERα protein and isolated ligand-binding domain, we found that these mutants promoted ERα dimerization, stability, and nuclear localization. Point mutations that selectively disrupted dimerization abrogated E2-independent transcriptional activity of these dimer-promoting mutants. The results reveal a distinct mechanism for activation of ERα function through enforced receptor dimerization and suggest dimer disruption as a potential therapeutic strategy to treat ER-dependent cancers.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Dimerização , Estradiol/farmacologia , Estradiol/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Ligantes , MutaçãoRESUMO
OBJECTIVE: To systematically assess the Canadian federal government's current alcohol policies in relation to public health best practices. METHODS: The 2022 Canadian Alcohol Policy Evaluation (CAPE) Project assessed federal alcohol policies across 10 domains. Policy domains were weighted according to evidence for their relative impact, including effectiveness and scope. A detailed scoring rubric of best practices was developed and externally reviewed by international experts. Policy data were collected between June and December 2022, using official legislation, government websites, and data sources identified from previous iterations of CAPE as sources. Contacts within relevant government departments provided any additional data sources, reviewed the accuracy and completeness of the data, and provided amendments as needed. Data were scored independently by members of the research team. Final policy scores were tabulated and presented as a weighted overall average score and as unweighted domain-specific scores. RESULTS: Compared to public health best practices, the federal government of Canada scored 37% overall. The three most impactful domains-(1) pricing and taxation, (2) marketing and advertising controls, and (3) impaired driving countermeasures-received some of the lowest scores (39%, 10%, and 40%, respectively). Domain-specific scores varied considerably from 0% for minimum legal age policies to 100% for controls on physical availability of alcohol. CONCLUSION: Many evidence-informed alcohol policies have not been adopted, or been adopted only partially, by the Canadian federal government. Urgent adoption of the recommended policies is needed to prevent and reduce the enormous health, social, and economic costs of alcohol use in Canada.
RéSUMé: OBJECTIF: Évaluer de manière systématique les politiques sur l'alcool actuelles du gouvernement fédéral canadien dans le cadre de pratiques de santé publique exemplaires. MéTHODES: Le projet de l'Évaluation des politiques canadiennes sur l'alcoolâ¯2022 a évalué les politiques fédérales sur l'alcool dans dix domaines. Ces domaines de politiques ont été pondérés en fonction de preuves sur leurs répercussions relatives, notamment leur efficacité et leur portée. Une échelle d'évaluation descriptive détaillée de pratiques exemplaires a été élaborée et examinée à l'externe. Entre juin et décembre 2022, des données sur les politiques ont été recueillies dans la législation officielle, sur des sites Web du gouvernement et au moyen de sources identifiées comme telles au cours des itérations précédentes du projet de l'Évaluation des politiques canadiennes sur l'alcool. Des personnes-ressources au sein des ministères concernés ont communiqué d'autres sources de données, examiné l'exactitude et le caractère exhaustif de ces données et apporté les modifications nécessaires. Les données ont été évaluées indépendamment par des membres de l'équipe de recherche. Les scores de politiques finaux ont été inscrits dans des tableaux et présentés sous forme d'une moyenne générale pondérée et de scores non pondérés par domaine. RéSULTATS: Comparativement aux pratiques de santé publique exemplaire, le gouvernement fédéral du Canada a obtenu un score général de 37 %. Les trois domaines susceptibles d'avoir les plus grandes répercussions, à savoir 1) la fixation des prix et la taxation, 2) le contrôle du marketing et de la publicité, et 3) les mesures contre la conduite avec facultés affaiblies, se sont vu attribuer parmi les scores les plus bas (39 %, 10 %, et 40 % respectivement). Les scores par domaine variaient considérablement, allant de 0 % pour les politiques sur l'âge minimum légal à 100 % pour le contrôle de la disponibilité physique de l'alcool. CONCLUSION: De nombreuses politiques sur l'alcool reposant sur des preuves n'ont pas été adoptées, ou l'ont été seulement partiellement, par le gouvernement fédéral canadien. Il est urgent d'appliquer les politiques recommandées pour prévenir et réduire les énormes coûts sanitaires, sociaux et économiques de la consommation d'alcool au Canada.
Assuntos
Consumo de Bebidas Alcoólicas , Política de Saúde , Canadá , Humanos , Consumo de Bebidas Alcoólicas/legislação & jurisprudência , Consumo de Bebidas Alcoólicas/prevenção & controle , Governo Federal , Política Pública , Bebidas Alcoólicas/economia , Prioridades em SaúdeRESUMO
Expanded CAG/CTG repeats are sites of DNA damage, leading to repeat length changes. Homologous recombination (HR) is one cause of repeat instability and we hypothesized that gap filling was a driver of repeat instability during HR. To test this, we developed an assay such that resection and ssDNA gap fill-in would occur across a (CAG)70 or (CTG)70 repeat tract. When the ssDNA template was a CTG sequence, there were increased repeat contractions and a fragile site was created leading to large-scale deletions. When the CTG sequence was on the resected strand, resection was inhibited, resulting in repeat expansions. Increased nucleolytic processing by deletion of Rad9, the ortholog of 53BP1, rescued repeat instability and chromosome breakage. Loss of Rad51 increased contractions implicating a protective role for Rad51 on ssDNA. Together, our work implicates structure-forming repeats as an impediment to resection and gap-filling which can lead to mutations and large-scale deletions.
Assuntos
Quebra Cromossômica , Dano ao DNA , Humanos , Mutação , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
The FOXA1 pioneer factor is an essential mediator of steroid receptor function in multiple hormone-dependent cancers, including breast and prostate cancers, enabling nuclear receptors such as estrogen receptor (ER) and androgen receptor (AR) to activate lineage-specific growth programs. FOXA1 is also highly expressed in non-small cell lung cancer (NSCLC), but whether and how it regulates tumor growth in this context is not known. Analyzing data from loss-of-function screens, we identified a subset of NSCLC tumor lines where proliferation is FOXA1 dependent. Using rapid immunoprecipitation and mass spectrometry of endogenous protein, we identified chromatin-localized interactions between FOXA1 and glucocorticoid receptor (GR) in these tumor cells. Knockdown of GR inhibited proliferation of FOXA1-dependent, but not FOXA1-independent NSCLC cells. In these FOXA1-dependent models, FOXA1 and GR cooperate to regulate gene targets involved in EGF signaling and G1-S cell-cycle progression. To investigate the therapeutic potential for targeting this complex, we examined the effects of highly selective inhibitors of the GR ligand-binding pocket and found that GR antagonism with ORIC-101 suppressed FOXA1/GR target expression, activation of EGF signaling, entry into the S-phase, and attendant proliferation in vitro and in vivo. Taken together, our findings point to a subset of NSCLCs harboring a dependence on the FOXA1/GR growth program and provide rationale for its therapeutic targeting. Significance: NSCLC is the leading cause of cancer deaths worldwide. There is a need to identify novel druggable dependencies. We identify a subset of NSCLCs dependent on FOXA1-GR and sensitive to GR antagonism.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Fator 3-alfa Nuclear de Hepatócito , Neoplasias Pulmonares , Receptores de Glucocorticoides , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Fator de Crescimento Epidérmico , Neoplasias Pulmonares/tratamento farmacológico , Receptores de Glucocorticoides/genética , Fator 3-alfa Nuclear de Hepatócito/genéticaRESUMO
The most prominent homozygous deletions in cancer affect chromosome 9p21.3 and eliminate CDKN2A/B tumor suppressors, disabling a cell-intrinsic barrier to tumorigenesis. Half of 9p21.3 deletions, however, also encompass a type I interferon (IFN) gene cluster; the consequences of this co-deletion remain unexplored. To functionally dissect 9p21.3 and other large genomic deletions, we developed a flexible deletion engineering strategy, MACHETE (molecular alteration of chromosomes with engineered tandem elements). Applying MACHETE to a syngeneic mouse model of pancreatic cancer, we found that co-deletion of the IFN cluster promoted immune evasion, metastasis and immunotherapy resistance. Mechanistically, IFN co-deletion disrupted type I IFN signaling in the tumor microenvironment, leading to marked changes in infiltrating immune cells and escape from CD8+ T-cell surveillance, effects largely driven by the poorly understood interferon epsilon. These results reveal a chromosomal deletion that disables both cell-intrinsic and cell-extrinsic tumor suppression and provide a framework for interrogating large deletions in cancer and beyond.
Assuntos
Interferons , Neoplasias , Animais , Camundongos , Deleção Cromossômica , Cromossomos , Evasão da Resposta Imune , Microambiente Tumoral/genética , Sequências de Repetição em TandemRESUMO
SMARCA4/BRG1 encodes for one of two mutually exclusive ATPases present in mammalian SWI/SNF chromatin remodeling complexes and is frequently mutated in human lung adenocarcinoma. However, the functional consequences of SMARCA4 mutation on tumor initiation, progression, and chromatin regulation in lung cancer remain poorly understood. Here, we demonstrate that loss of Smarca4 sensitizes club cell secretory protein-positive cells within the lung in a cell type-dependent fashion to malignant transformation and tumor progression, resulting in highly advanced dedifferentiated tumors and increased metastatic incidence. Consistent with these phenotypes, Smarca4-deficient primary tumors lack lung lineage transcription factor activities and resemble a metastatic cell state. Mechanistically, we show that Smarca4 loss impairs the function of all three classes of SWI/SNF complexes, resulting in decreased chromatin accessibility at lung lineage motifs and ultimately accelerating tumor progression. Thus, we propose that the SWI/SNF complex via Smarca4 acts as a gatekeeper for lineage-specific cellular transformation and metastasis during lung cancer evolution. SIGNIFICANCE: We demonstrate cell-type specificity in the tumor-suppressive functions of SMARCA4 in the lung, pointing toward a critical role of the cell-of-origin in driving SWI/SNF-mutant lung adenocarcinoma. We further show the direct effects of SMARCA4 loss on SWI/SNF function and chromatin regulation that cause aggressive malignancy during lung cancer evolution.This article is highlighted in the In This Issue feature, p. 275.
Assuntos
Adenocarcinoma de Pulmão/genética , Transformação Celular Neoplásica , DNA Helicases/genética , Neoplasias Pulmonares/genética , Metástase Neoplásica , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adenocarcinoma de Pulmão/secundário , Animais , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/patologia , CamundongosRESUMO
Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(-) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.
RESUMO
Cyclin-dependent kinases 4 and 6 (CDK4/6) represent a major therapeutic vulnerability for breast cancer. The kinases are clinically targeted via ATP competitive inhibitors (CDK4/6i); however, drug resistance commonly emerges over time. To understand CDK4/6i resistance, we surveyed over 1,300 breast cancers and identified several genetic alterations (e.g., FAT1, PTEN, or ARID1A loss) converging on upregulation of CDK6. Mechanistically, we demonstrate CDK6 causes resistance by inducing and binding CDK inhibitor INK4 proteins (e.g., p18INK4C). In vitro binding and kinase assays together with physical modeling reveal that the p18INK4C-cyclin D-CDK6 complex occludes CDK4/6i binding while only weakly suppressing ATP binding. Suppression of INK4 expression or its binding to CDK6 restores CDK4/6i sensitivity. To overcome this constraint, we developed bifunctional degraders conjugating palbociclib with E3 ligands. Two resulting lead compounds potently degraded CDK4/6, leading to substantial antitumor effects in vivo, demonstrating the promising therapeutic potential for retargeting CDK4/6 despite CDK4/6i resistance. SIGNIFICANCE: CDK4/6 kinase activation represents a common mechanism by which oncogenic signaling induces proliferation and is potentially targetable by ATP competitive inhibitors. We identify a CDK6-INK4 complex that is resilient to current-generation inhibitors and develop a new strategy for more effective inhibition of CDK4/6 kinases.This article is highlighted in the In This Issue feature, p. 275.
Assuntos
Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Proteínas Inibidoras de Quinase Dependente de Ciclina/química , Resistencia a Medicamentos Antineoplásicos , Piperazinas/química , Inibidores de Proteínas Quinases/química , Piridinas/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proteínas Inibidoras de Quinase Dependente de Ciclina/administração & dosagem , Proteínas Inibidoras de Quinase Dependente de Ciclina/uso terapêutico , Feminino , Humanos , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Proteínas Supressoras de Tumor/metabolismoRESUMO
Single-cell ATAC sequencing using combinatorial indexing (sciATAC-seq) enables the identification of chromatin accessibility profiles at single-cell resolution with a dual-barcoding approach during transposition and library construction. Unlike commercial droplet-based approaches, sciATAC-seq is a cost-effective, extensible strategy that permits flexibility in the experimental scale via multiplexed barcoding across samples or perturbations. In contrast, droplet-based approaches have higher cell recovery and may be advantageous when cell input is limited. The step-by-step sciATAC-seq protocol described here is amenable to diverse cell types and fixed samples. For complete details on the use and execution of this protocol, please refer to LaFave et al. (2020).
Assuntos
Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/genética , Análise de Célula Única/métodos , Animais , Biologia Computacional/métodos , Epigênese Genética , CamundongosRESUMO
Parkinson's Disease (PD) is symptomatically managed with L-DOPA but chronic use results in L-DOPA-induced dyskinesia (LID) characterized by abnormal involuntary movements (AIMs). In LID, dopamine D3 receptors (D3R) are upregulated on D1 receptor (D1R)-bearing medium spiny neurons where the can synergistically drive downstream signaling and motor behaviors. Despite evidence implying D1R-D3R cooperativity in LID, the dyskinesiogenic role of D3R has never been directly tested. To this end, we developed a specific cre-dependent microRNA (miRNA) to irreversibly prevent D3R upregulation in D1R striatal cells. D1-Cre rats received unilateral 6-hydroxydopamine lesions. Three weeks later, rats received an adeno-associated virus expressing either D3R miRNA or a scrambled (SCR) miRNA delivered into the striatum. After 4 weeks, rats received chronic L-DOPA (6 mg/kg) or vehicle. AIMs development and motor behaviors were assayed throughout treatment. At the conclusion of the experiment, efficacy and fidelity of the miRNA strategy was analyzed using in situ hybridization (ISH). ISH analyses demonstrated that D1R+/D3R+ cells were upregulated in LID and that the selective D3R miRNA reduced D1R+/D3R+ co-expression. Importantly, silencing of D3R also significantly attenuated LID development without impacting L-DOPA efficacy or other locomotion. These data highlight a dyskinesiogenic role of D3R within D1R cells in LID and highlight aberrant D1R-D3R interactions as targets of LID management.
Assuntos
Dopaminérgicos/efeitos adversos , Discinesia Induzida por Medicamentos/genética , Discinesia Induzida por Medicamentos/prevenção & controle , Levodopa/efeitos adversos , Neostriado/patologia , Receptores de Dopamina D1/genética , Receptores de Dopamina D3/genética , Animais , Comportamento Animal , Discinesia Induzida por Medicamentos/psicologia , Feminino , Terapia Genética , Hidroxidopaminas , Masculino , MicroRNAs/genética , Neostriado/metabolismo , Desempenho Psicomotor , RatosRESUMO
Regulatory networks that maintain functional, differentiated cell states are often dysregulated in tumor development. Here, we use single-cell epigenomics to profile chromatin state transitions in a mouse model of lung adenocarcinoma (LUAD). We identify an epigenomic continuum representing loss of cellular identity and progression toward a metastatic state. We define co-accessible regulatory programs and infer key activating and repressive chromatin regulators of these cell states. Among these co-accessibility programs, we identify a pre-metastatic transition, characterized by activation of RUNX transcription factors, which mediates extracellular matrix remodeling to promote metastasis and is predictive of survival across human LUAD patients. Together, these results demonstrate the power of single-cell epigenomics to identify regulatory programs to uncover mechanisms and key biomarkers of tumor progression.