RESUMO
Glycine-12 mutations in the GTPase KRAS (KRASG12) are an initiating event for development of lung adenocarcinoma (LUAD). KRASG12 mutations promote cell-intrinsic rewiring of alveolar type-II progenitor (AT2) cells, but to what extent such changes interplay with lung homeostasis and cell fate pathways is unclear. Here, we generated single-cell RNA-seq (scRNA-seq) profiles from AT2-mesenchyme organoid co-cultures, mice, and stage-IA LUAD patients, identifying conserved regulators of AT2 transcriptional dynamics and defining the impact of KRASG12D mutation with temporal resolution. In AT2WT organoids, we found a transient injury/plasticity state preceding AT2 self-renewal and AT1 differentiation. Early-stage AT2KRAS cells exhibited perturbed gene expression dynamics, most notably retention of the injury/plasticity state. The injury state in AT2KRAS cells of patients, mice, and organoids was distinguishable from AT2WT states via altered receptor expression, including co-expression of ITGA3 and SRC. The combination of clinically relevant KRASG12D and SRC inhibitors impaired AT2KRAS organoid growth. Together, our data show that an injury/plasticity state essential for lung repair is co-opted during AT2 self-renewal and LUAD initiation, suggesting that early-stage LUAD may be susceptible to interventions that target specifically the oncogenic nature of this cell state.
Assuntos
Neoplasias Pulmonares , Organoides , Proteínas Proto-Oncogênicas p21(ras) , Animais , Humanos , Camundongos , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Adenocarcinoma de Pulmão/metabolismo , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Mutação , Organoides/metabolismo , Organoides/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases da Família src/metabolismo , Quinases da Família src/genéticaRESUMO
Acute injury in the airways or the lung activates local progenitors and stimulates changes in cell-cell interactions to restore homeostasis, but it is not appreciated how more distant niches are impacted. We utilized mouse models of airway-specific epithelial injury to examine secondary tissue-wide alveolar, immune, and mesenchymal responses. Single-cell transcriptomics and in vivo validation revealed transient, tissue-wide proliferation of alveolar type 2 (AT2) progenitor cells after club cell-specific ablation. The AT2 cell proliferative response was reliant on alveolar macrophages (AMs) via upregulation of Spp1 which encodes the secreted factor Osteopontin. A previously uncharacterized mesenchymal population we termed Mesenchymal Airway/Adventitial Niche Cell 2 (MANC2) also exhibited dynamic changes in abundance and a pro-fibrotic transcriptional signature after club cell ablation in an AM-dependent manner. Overall, these results demonstrate that acute airway damage can trigger distal lung responses including altered cell-cell interactions that may contribute to potential vulnerabilities for further dysregulation and disease.
RESUMO
The lung contains multiple progenitor cell types, but how their responses are choreographed during injury repair and whether this changes with age is poorly understood. We report that histone H3 lysine 9 di-methylation (H3K9me2), mediated by the methyltransferase G9a, regulates the dynamics of distal lung epithelial progenitor cells and that this regulation deteriorates with age. In aged mouse lungs, H3K9me2 loss coincided with fewer alveolar type 2 (AT2) cell progenitors and reduced alveolar regeneration but increased the frequency and activity of multipotent bronchioalveolar stem cells (BASCs) and bronchiolar progenitor club cells. H3K9me2 depletion in young mice decreased AT2 progenitor activity and impaired alveolar injury repair. Conversely, H3K9me2 depletion increased chromatin accessibility of bronchiolar cell genes, increased BASC frequency, and accelerated bronchiolar cell injury repair. These findings indicate that during aging, the epigenetic regulation that coordinates lung progenitor cells' regenerative responses becomes dysregulated, aiding our understanding of age-related susceptibility to lung disease.
Assuntos
Epigênese Genética , Pulmão , Camundongos , Animais , Pulmão/metabolismo , Cromatina/metabolismo , Metilação , Processamento de Proteína Pós-TraducionalRESUMO
Telomeric C-rich repeated DNA sequences fold into tetrahelical i-motif structures in vitro at acidic pH. While studies have suggested that i-motifs may form in cells, little is known about their potential role in human telomere biology. In this study, we explore the effect of telomeric C-strands and i-motifs on the ability of human telomerase to extend G-rich substrates. To promote i-motif formation at neutral pH, we use telomeric sequences where the cytidines have been substituted with 2'-fluoroarabinocytidine. Using FRET-based studies, we show that the stabilized i-motifs resist hybridization to concomitant parallel G-quadruplexes, implying that both structures could exist simultaneously at telomeric termini. Moreover, through telomerase activity assays, we show that both unstructured telomeric C-strands and telomeric i-motifs can inhibit the activity and processivity of telomerase extension of parallel G-quadruplexes and linear telomeric DNA. The data suggest at least three modes of inhibition by C-strands and i-motifs: direct hybridization to the substrate DNA, hybridization to nascent product DNA resulting in early telomerase dissociation, and interference with the unique mechanism of telomerase unwinding and extension of a G-quadruplex. Overall, this study highlights a potential inhibitory role for the telomeric C-strand in telomere maintenance.
Assuntos
Quadruplex G , Telomerase , Humanos , Telomerase/metabolismo , DNA/química , Hibridização de Ácido Nucleico , Telômero/metabolismoRESUMO
Alveolar type 2 (AT2) cells, the epithelial progenitor cells of the distal lung, are known to be the prominent cell of origin for lung adenocarcinoma. The regulatory programs that control chromatin and gene expression in AT2 cells during the early stages of tumor initiation are not well understood. Here, we dissected the response of AT2 cells to Kras activation and p53 loss (KP) using combined single cell RNA and ATAC sequencing in an established tumor organoid system. Multi-omic analysis showed that KP tumor organoid cells exhibit two major cellular states: one more closely resembling AT2 cells (SPC-high) and another with loss of AT2 identity (hereafter, Hmga2-high). These cell states are characterized by unique transcription factor (TF) networks, with SPC-high states associated with TFs known to regulate AT2 cell fate during development and homeostasis, and distinct TFs associated with the Hmga2-high state. CD44 was identified as a marker of the Hmga2-high state, and was used to separate organoid cultures for functional comparison of these two cell states. Organoid assays and orthotopic transplantation studies indicated that SPC-high cells have higher tumorigenic capacity in the lung microenvironment compared to Hmga2-high cells. These findings highlight the utility of understanding chromatin regulation in the early oncogenic versions of epithelial cells, which may reveal more effective means to intervene the progression of Kras-driven lung cancer.
RESUMO
Lung progenitor cells are crucial for regeneration following injury, yet it is unclear whether lung progenitor cells can be functionally engrafted after transplantation. We transplanted organoid cells derived from alveolar type II (AT2) cells enriched by SCA1-negative status (SNO) or multipotent SCA1-positive progenitor cells (SPO) into injured mouse lungs. Transplanted SNO cells are retained in the alveolar regions, whereas SPO cells incorporate into airway and alveolar regions. Single-cell transcriptomics demonstrate that transplanted SNO cells are comparable to native AT2 cells. Transplanted SPO cells exhibit transcriptional hallmarks of alveolar and airway cells, as well as transitional cell states identified in disease. Transplanted cells proliferate after re-injury of recipient mice and retain organoid-forming capacity. Thus, lung epithelial organoid cells exhibit progenitor cell functions after reintroduction to the lung. This study reveals methods to interrogate lung progenitor cell potential and model transitional cell states relevant to pathogenic features of lung disease in vivo.
Assuntos
Organoides , Ataxias Espinocerebelares , Animais , Diferenciação Celular , Células Epiteliais , Pulmão , Camundongos , Células-TroncoRESUMO
Lung cancer in never smokers (LCINS) is a common cause of cancer mortality but its genomic landscape is poorly characterized. Here high-coverage whole-genome sequencing of 232 LCINS showed 3 subtypes defined by copy number aberrations. The dominant subtype (piano), which is rare in lung cancer in smokers, features somatic UBA1 mutations, germline AR variants and stem cell-like properties, including low mutational burden, high intratumor heterogeneity, long telomeres, frequent KRAS mutations and slow growth, as suggested by the occurrence of cancer drivers' progenitor cells many years before tumor diagnosis. The other subtypes are characterized by specific amplifications and EGFR mutations (mezzo-forte) and whole-genome doubling (forte). No strong tobacco smoking signatures were detected, even in cases with exposure to secondhand tobacco smoke. Genes within the receptor tyrosine kinase-Ras pathway had distinct impacts on survival; five genomic alterations independently doubled mortality. These findings create avenues for personalized treatment in LCINS.
Assuntos
Variações do Número de Cópias de DNA/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , não Fumantes/estatística & dados numéricos , Adulto , Idoso , Idoso de 80 Anos ou mais , Receptores ErbB/genética , Feminino , Genoma/genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores Androgênicos/genética , Fatores de Risco , Fumar/genética , Enzimas Ativadoras de Ubiquitina/genética , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
Mutant KRAS is a common driver in epithelial cancers. Nevertheless, molecular changes occurring early after activation of oncogenic KRAS in epithelial cells remain poorly understood. We compared transcriptional changes at single-cell resolution after KRAS activation in four sample sets. In addition to patient samples and genetically engineered mouse models, we developed organoid systems from primary mouse and human induced pluripotent stem cell-derived lung epithelial cells to model early-stage lung adenocarcinoma. In all four settings, alveolar epithelial progenitor (AT2) cells expressing oncogenic KRAS had reduced expression of mature lineage identity genes. These findings demonstrate the utility of our in vitro organoid approaches for uncovering the early consequences of oncogenic KRAS expression. This resource provides an extensive collection of datasets and describes organoid tools to study the transcriptional and proteomic changes that distinguish normal epithelial progenitor cells from early-stage lung cancer, facilitating the search for targets for KRAS-driven tumors.
Assuntos
Células-Tronco Pluripotentes Induzidas , Organoides , Animais , Humanos , Pulmão , Camundongos , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Human genome function is underpinned by the primary storage of genetic information in canonical B-form DNA, with a second layer of DNA structure providing regulatory control. I-motif structures are thought to form in cytosine-rich regions of the genome and to have regulatory functions; however, in vivo evidence for the existence of such structures has so far remained elusive. Here we report the generation and characterization of an antibody fragment (iMab) that recognizes i-motif structures with high selectivity and affinity, enabling the detection of i-motifs in the nuclei of human cells. We demonstrate that the in vivo formation of such structures is cell-cycle and pH dependent. Furthermore, we provide evidence that i-motif structures are formed in regulatory regions of the human genome, including promoters and telomeric regions. Our results support the notion that i-motif structures provide key regulatory roles in the genome.
Assuntos
Núcleo Celular/química , DNA/química , Conformação de Ácido Nucleico , Ciclo Celular , DNA/imunologia , Genoma Humano , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Fragmentos de Imunoglobulinas/imunologia , Células MCF-7 , Regiões Promotoras Genéticas , TelômeroRESUMO
Cancer is a multifactorial disease driven by a combination of genetic and environmental factors. Many cancer driver mutations have been characterised in protein-coding regions of the genome. However, mutations in noncoding regions associated with cancer have been less investigated. G-quadruplex (G4) nucleic acids are four-stranded secondary structures formed in guanine-rich sequences and prevalent in the regulatory regions. In this study, we used published whole cancer genome sequence data to find mutations in cancer patients that overlap potential RNA G4-forming sequences in 5' UTRs. Using RNAfold, we assessed the effect of these mutations on the thermodynamic stability of predicted RNA G4s in the context of full-length 5' UTRs. Of the 217 identified mutations, we found that 33 are predicted to destabilise and 21 predicted to stabilise potential RNA G4s. We experimentally validated the effect of destabilising mutations in the 5' UTRs of BCL2 and CXCL14 and one stabilising mutation in the 5' UTR of TAOK2. These mutations resulted in an increase or a decrease in translation of these mRNAs, respectively. These findings suggest that mutations that modulate the G4 stability in the noncoding regions could act as cancer driver mutations, which present an opportunity for early cancer diagnosis using individual sequencing information.
Assuntos
Quadruplex G , Regulação da Expressão Gênica , Mutação , Neoplasias/genética , RNA/química , RNA/genética , Regiões não Traduzidas , Regiões 5' não Traduzidas , Linhagem Celular Tumoral , Quimiocinas CXC/genética , Genes Reporter , Genes bcl-2 , Humanos , Mutação Puntual , Estabilidade de RNA , TermodinâmicaRESUMO
It has been hypothesized that G-quadruplexes can sequester the 3' end of the telomere and prevent it from being extended by telomerase. Here we purify and characterize stable, conformationally homogenous human telomeric G-quadruplexes, and demonstrate that human telomerase is able to extend parallel, intermolecular conformations in vitro. These G-quadruplexes align correctly with the RNA template of telomerase, demonstrating that at least partial G-quadruplex resolution is required. A highly purified preparation of human telomerase retains this extension ability, establishing that the core telomerase enzyme complex is sufficient for partial G-quadruplex resolution and extension. The parallel-specific G-quadruplex ligand N-methyl mesoporphyrin IX (NMM) causes an increase in telomeric G-quadruplexes, and we show that telomerase colocalizes with a subset of telomeric G-quadruplexes in vivo. The ability of telomerase to partially unwind, extend and localize to these structures implies that parallel telomeric G-quadruplexes may play an important biological role.
Assuntos
DNA/metabolismo , Quadruplex G , Telomerase/metabolismo , Western Blotting , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Quadruplex G/efeitos dos fármacos , Células HEK293 , Humanos , Hibridização in Situ Fluorescente , Mesoporfirinas/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Homeostase do TelômeroRESUMO
The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from syndromes such as dyskeratosis congenita, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic and thermodynamic analyses of wild-type telomerase and two disease-associated mutations in the reverse transcriptase domain. Differences in dissociation rates between primers with different 3' ends were independent of DNA affinities, revealing that initial binding of telomerase to telomeric DNA occurs through a previously undescribed two-step mechanism involving enzyme conformational changes. Both mutations affected DNA binding, but through different mechanisms: P704S specifically affected protein conformational changes during DNA binding, whereas R865H showed defects in binding to the 3' region of the DNA. To gain further insight at the structural level, we generated the first homology model of the human telomerase reverse transcriptase domain; the positions of P704S and R865H corroborate their observed mechanistic defects, providing validation for the structural model. Our data reveal the importance of protein interactions with the 3' end of telomeric DNA and the role of protein conformational change in telomerase DNA binding, and highlight naturally occurring disease mutations as a rich source of mechanistic insight.