RESUMO
Brain metastasis (BrM) is the most common form of brain cancer, characterized by neurologic disability and an abysmal prognosis. Unfortunately, our understanding of the biology underlying human BrMs remains rudimentary. Here, we present an integrative analysis of >100,000 malignant and non-malignant cells from 15 human parenchymal BrMs, generated by single-cell transcriptomics, mass cytometry, and complemented with mouse model- and in silico approaches. We interrogated the composition of BrM niches, molecularly defined the blood-tumor interface, and revealed stromal immunosuppressive states enriched with infiltrated T cells and macrophages. Specific single-cell interrogation of metastatic tumor cells provides a framework of 8 functional cell programs that coexist or anticorrelate. Collectively, these programs delineate two functional BrM archetypes, one proliferative and the other inflammatory, that are evidently shaped through tumor-immune interactions. Our resource provides a foundation to understand the molecular basis of BrM in patients with tumor cell-intrinsic and host environmental traits.
Assuntos
Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Adulto , Idoso , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/imunologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Variação Genética , Humanos , Evasão da Resposta Imune , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Modelos Biológicos , Células Mieloides/patologia , Análise de Componente Principal , RNA-Seq , Análise de Célula Única , Linfócitos T/imunologiaRESUMO
DNA has not been utilized to record temporal information, although DNA has been used to record biological information and to compute mathematical problems. Here, we found that indel generation by Cas9 and guide RNA can occur at steady rates, in contrast to typical dynamic biological reactions, and the accumulated indel frequency can be a function of time. By measuring indel frequencies, we developed a method for recording and measuring absolute time periods over hours to weeks in mammalian cells. These time-recordings were conducted in several cell types, with different promoters and delivery vectors for Cas9, and in both cultured cells and cells of living mice. As applications, we recorded the duration of chemical exposure and the lengths of elapsed time since the onset of biological events (e.g., heat exposure and inflammation). We propose that our systems could serve as synthetic "DNA clocks."
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Animais , Sequência de Bases , Microambiente Celular , Simulação por Computador , Células HEK293 , Meia-Vida , Humanos , Mutação INDEL/genética , Inflamação/patologia , Integrases/metabolismo , Masculino , Camundongos Nus , Regiões Promotoras Genéticas/genética , RNA Guia de Cinetoplastídeos/genética , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
Despite considerable efforts, the mechanisms linking genomic alterations to the transcriptional identity of cancer cells remain elusive. Integrative genomic analysis, using a network-based approach, identified 407 master regulator (MR) proteins responsible for canalizing the genetics of individual samples from 20 cohorts in The Cancer Genome Atlas (TCGA) into 112 transcriptionally distinct tumor subtypes. MR proteins could be further organized into 24 pan-cancer, master regulator block modules (MRBs), each regulating key cancer hallmarks and predictive of patient outcome in multiple cohorts. Of all somatic alterations detected in each individual sample, >50% were predicted to induce aberrant MR activity, yielding insight into mechanisms linking tumor genetics and transcriptional identity and establishing non-oncogene dependencies. Genetic and pharmacological validation assays confirmed the predicted effect of upstream mutations and MR activity on downstream cellular identity and phenotype. Thus, co-analysis of mutational and gene expression profiles identified elusive subtypes and provided testable hypothesis for mechanisms mediating the effect of genetic alterations.
Assuntos
Neoplasias/genética , Transcrição Gênica , Adenocarcinoma/genética , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Genoma Humano , Células HEK293 , Humanos , Camundongos Nus , Mutação/genética , Reprodutibilidade dos TestesRESUMO
Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable success in hematological malignancies but remains ineffective in solid tumors, due in part to CAR T cell exhaustion in the solid tumor microenvironment. To study dysfunction of mesothelin-redirected CAR T cells in pancreatic cancer, we establish a robust model of continuous antigen exposure that recapitulates hallmark features of T cell exhaustion and discover, both in vitro and in CAR T cell patients, that CAR dysregulation is associated with a CD8+ T-to-NK-like T cell transition. Furthermore, we identify a gene signature defining CAR and TCR dysregulation and transcription factors, including SOX4 and ID3 as key regulators of CAR T cell exhaustion. Our findings shed light on the plasticity of human CAR T cells and demonstrate that genetic downmodulation of ID3 and SOX4 expression can improve the efficacy of CAR T cell therapy in solid tumors by preventing or delaying CAR T cell dysfunction.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia Adotiva/métodos , Neoplasias Pancreáticas/terapia , Receptores de Antígenos Quiméricos/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas Inibidoras de Diferenciação/imunologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Proteínas de Neoplasias/imunologia , Fatores de Transcrição SOXC/imunologiaRESUMO
Metastasis has been considered as the terminal step of tumor progression. However, recent genomic studies suggest that many metastases are initiated by further spread of other metastases. Nevertheless, the corresponding pre-clinical models are lacking, and underlying mechanisms are elusive. Using several approaches, including parabiosis and an evolving barcode system, we demonstrated that the bone microenvironment facilitates breast and prostate cancer cells to further metastasize and establish multi-organ secondary metastases. We uncovered that this metastasis-promoting effect is driven by epigenetic reprogramming that confers stem cell-like properties on cancer cells disseminated from bone lesions. Furthermore, we discovered that enhanced EZH2 activity mediates the increased stemness and metastasis capacity. The same findings also apply to single cell-derived populations, indicating mechanisms distinct from clonal selection. Taken together, our work revealed an unappreciated role of the bone microenvironment in metastasis evolution and elucidated an epigenomic reprogramming process driving terminal-stage, multi-organ metastases.
Assuntos
Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Metástase Neoplásica , Neoplasias da Próstata/patologia , Microambiente Tumoral , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Impairment of protein phosphatases, including the family of serine/threonine phosphatases designated PP2A, is essential for the pathogenesis of many diseases, including cancer. The ability of PP2A to dephosphorylate hundreds of proteins is regulated by over 40 specificity-determining regulatory "B" subunits that compete for assembly and activation of heterogeneous PP2A heterotrimers. Here, we reveal how a small molecule, DT-061, specifically stabilizes the B56α-PP2A holoenzyme in a fully assembled, active state to dephosphorylate selective substrates, such as its well-known oncogenic target, c-Myc. Our 3.6 Å structure identifies molecular interactions between DT-061 and all three PP2A subunits that prevent dissociation of the active enzyme and highlight inherent mechanisms of PP2A complex assembly. Thus, our findings provide fundamental insights into PP2A complex assembly and regulation, identify a unique interfacial stabilizing mode of action for therapeutic targeting, and aid in the development of phosphatase-based therapeutics tailored against disease specific phospho-protein targets.
Assuntos
Proteína Fosfatase 2/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Ativadores de Enzimas/metabolismo , Células HEK293 , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteína Fosfatase 2/química , Subunidades ProteicasRESUMO
Glioblastomas exhibit vast inter- and intra-tumoral heterogeneity, complicating the development of effective therapeutic strategies. Current in vitro models are limited in preserving the cellular and mutational diversity of parental tumors and require a prolonged generation time. Here, we report methods for generating and biobanking patient-derived glioblastoma organoids (GBOs) that recapitulate the histological features, cellular diversity, gene expression, and mutational profiles of their corresponding parental tumors. GBOs can be generated quickly with high reliability and exhibit rapid, aggressive infiltration when transplanted into adult rodent brains. We further demonstrate the utility of GBOs to test personalized therapies by correlating GBO mutational profiles with responses to specific drugs and by modeling chimeric antigen receptor T cell immunotherapy. Our studies show that GBOs maintain many key features of glioblastomas and can be rapidly deployed to investigate patient-specific treatment strategies. Additionally, our live biobank establishes a rich resource for basic and translational glioblastoma research.
Assuntos
Técnicas de Cultura de Células/métodos , Glioblastoma/metabolismo , Organoides/crescimento & desenvolvimento , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Bancos de Espécimes Biológicos , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Modelos Biológicos , Organoides/metabolismo , Reprodutibilidade dos Testes , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
It has generally proven challenging to produce functional ß cells in vitro. Here, we describe a previously unidentified protein C receptor positive (Procr+) cell population in adult mouse pancreas through single-cell RNA sequencing (scRNA-seq). The cells reside in islets, do not express differentiation markers, and feature epithelial-to-mesenchymal transition characteristics. By genetic lineage tracing, Procr+ islet cells undergo clonal expansion and generate all four endocrine cell types during adult homeostasis. Sorted Procr+ cells, representing â¼1% of islet cells, can robustly form islet-like organoids when cultured at clonal density. Exponential expansion can be maintained over long periods by serial passaging, while differentiation can be induced at any time point in culture. ß cells dominate in differentiated islet organoids, while α, δ, and PP cells occur at lower frequencies. The organoids are glucose-responsive and insulin-secreting. Upon transplantation in diabetic mice, these organoids reverse disease. These findings demonstrate that the adult mouse pancreatic islet contains a population of Procr+ endocrine progenitors.
Assuntos
Técnicas de Cultura de Células/métodos , Receptor de Proteína C Endotelial/metabolismo , Ilhotas Pancreáticas/citologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Nus , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Proteína C/metabolismo , Células-Tronco/citologiaRESUMO
Oncogenic transformation is associated with profound changes in cellular metabolism, but whether tracking these can improve disease stratification or influence therapy decision-making is largely unknown. Using the iKnife to sample the aerosol of cauterized specimens, we demonstrate a new mode of real-time diagnosis, coupling metabolic phenotype to mutant PIK3CA genotype. Oncogenic PIK3CA results in an increase in arachidonic acid and a concomitant overproduction of eicosanoids, acting to promote cell proliferation beyond a cell-autonomous manner. Mechanistically, mutant PIK3CA drives a multimodal signaling network involving mTORC2-PKCζ-mediated activation of the calcium-dependent phospholipase A2 (cPLA2). Notably, inhibiting cPLA2 synergizes with fatty acid-free diet to restore immunogenicity and selectively reduce mutant PIK3CA-induced tumorigenicity. Besides highlighting the potential for metabolic phenotyping in stratified medicine, this study reveals an important role for activated PI3K signaling in regulating arachidonic acid metabolism, uncovering a targetable metabolic vulnerability that largely depends on dietary fat restriction. VIDEO ABSTRACT.
Assuntos
Ácido Araquidônico/análise , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Eicosanoides/metabolismo , Animais , Ácido Araquidônico/metabolismo , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/genética , Citosol/metabolismo , Eicosanoides/fisiologia , Ativação Enzimática , Feminino , Humanos , Metabolismo dos Lipídeos/fisiologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipases A2/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.
Assuntos
Anticorpos/uso terapêutico , Aprendizado Profundo , Diagnóstico por Computador/métodos , Quimioterapia Assistida por Computador/métodos , Neoplasias/patologia , Animais , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos SCID , Metástase Neoplásica , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Software , Microambiente TumoralRESUMO
The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Feminino , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Glicosilação , Células HEK293 , Células Hep G2 , Xenoenxertos , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução GenéticaRESUMO
Activating mutations in NRAS account for 20%-30% of melanoma, but despite decades of research and in contrast to BRAF, no effective anti-NRAS therapies have been forthcoming. Here, we identify a previously uncharacterized serine/threonine kinase STK19 as a novel NRAS activator. STK19 phosphorylates NRAS to enhance its binding to its downstream effectors and promotes oncogenic NRAS-mediated melanocyte malignant transformation. A recurrent D89N substitution in STK19 whose alterations were identified in 25% of human melanomas represents a gain-of-function mutation that interacts better with NRAS to enhance melanocyte transformation. STK19D89N knockin leads to skin hyperpigmentation and promotes NRASQ61R-driven melanomagenesis in vivo. Finally, we developed ZT-12-037-01 (1a) as a specific STK19-targeted inhibitor and showed that it effectively blocks oncogenic NRAS-driven melanocyte malignant transformation and melanoma growth in vitro and in vivo. Together, our findings provide a new and viable therapeutic strategy for melanomas harboring NRAS mutations.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Melanoma/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Feminino , Células HEK293 , Humanos , Melanócitos/metabolismo , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Mutação , Fosforilação , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genéticaRESUMO
Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.
Assuntos
Neoplasias da Mama/metabolismo , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Cinamatos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Antagonistas do Receptor de Estrogênio/uso terapêutico , Feminino , Fulvestranto/uso terapêutico , Células HEK293 , Xenoenxertos , Humanos , Indazóis/farmacologia , Ligantes , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Polimorfismo de Nucleotídeo Único , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Intrinsic and acquired drug resistance and induction of secondary malignancies limit successful chemotherapy. Because mutagenic translesion synthesis (TLS) contributes to chemoresistance as well as treatment-induced mutations, targeting TLS is an attractive avenue for improving chemotherapeutics. However, development of small molecules with high specificity and in vivo efficacy for mutagenic TLS has been challenging. Here, we report the discovery of a small-molecule inhibitor, JH-RE-06, that disrupts mutagenic TLS by preventing recruitment of mutagenic POL ζ. Remarkably, JH-RE-06 targets a nearly featureless surface of REV1 that interacts with the REV7 subunit of POL ζ. Binding of JH-RE-06 induces REV1 dimerization, which blocks the REV1-REV7 interaction and POL ζ recruitment. JH-RE-06 inhibits mutagenic TLS and enhances cisplatin-induced toxicity in cultured human and mouse cell lines. Co-administration of JH-RE-06 with cisplatin suppresses the growth of xenograft human melanomas in mice, establishing a framework for developing TLS inhibitors as a novel class of chemotherapy adjuvants.
Assuntos
Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Mutagênese/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Quinolinas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/efeitos adversos , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA , Feminino , Técnicas de Silenciamento de Genes , Humanos , Proteínas Mad2/metabolismo , Camundongos , Camundongos Nus , Camundongos Transgênicos , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Quinolinas/química , Quinolinas/farmacologia , Transfecção , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Blind mole rats (BMRs) are small rodents, characterized by an exceptionally long lifespan (>21 years) and resistance to both spontaneous and induced tumorigenesis. Here we report that cancer resistance in the BMR is mediated by retrotransposable elements (RTEs). Cells and tissues of BMRs express very low levels of DNA methyltransferase 1. Following cell hyperplasia, the BMR genome DNA loses methylation, resulting in the activation of RTEs. Upregulated RTEs form cytoplasmic RNA-DNA hybrids, which activate the cGAS-STING pathway to induce cell death. Although this mechanism is enhanced in the BMR, we show that it functions in mice and humans. We propose that RTEs were co-opted to serve as tumor suppressors that monitor cell proliferation and are activated in premalignant cells to trigger cell death via activation of the innate immune response. Activation of RTEs is a double-edged sword, serving as a tumor suppressor but contributing to aging in late life via the induction of sterile inflammation.
Assuntos
Elementos de DNA Transponíveis/imunologia , Imunidade Inata/imunologia , Ratos-Toupeira/imunologia , Neoplasias/imunologia , Animais , Carcinogênese/imunologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Células Cultivadas , DNA/imunologia , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Ratos , Transdução de Sinais/imunologiaRESUMO
Epithelial cells have an ability termed 'cell competition', which is an immune surveillance-like function that extrudes precancerous cells from the epithelial layer, leading to apoptosis and clearance. However, it remains unclear how epithelial cells recognize and extrude transformed cells. Here, we discovered that a PirB family protein, leukocyte immunoglobulin-like receptor B3 (LILRB3), which is expressed on non-transformed epithelial cells, recognizes major histocompatibility complex class I (MHC class I) that is highly expressed on transformed cells. MHC class I interaction with LILRB3 expressed on normal epithelial cells triggers an SHP2-ROCK2 pathway that generates a mechanical force to extrude transformed cells. Removal of transformed cells occurs independently of natural killer (NK) cell or CD8+ cytotoxic T cell-mediated activity. This is a new mechanism in that the immunological ligand-receptor system generates a mechanical force in non-immune epithelial cells to extrude precancerous cells in the same epithelial layer.
Assuntos
Antígenos CD/metabolismo , Apoptose , Competição entre as Células , Células Epiteliais/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Pulmonares/metabolismo , Lesões Pré-Cancerosas/metabolismo , Receptores Imunológicos/metabolismo , Animais , Antígenos CD/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Cães , Células Epiteliais/imunologia , Células Epiteliais/patologia , Células HaCaT , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Células Madin Darby de Rim Canino , Mecanotransdução Celular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/imunologia , Lesões Pré-Cancerosas/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Células RAW 264.7 , Receptores Imunológicos/genética , Estresse Mecânico , Quinases Associadas a rho/metabolismoRESUMO
The anatomic location and immunologic characteristics of brain tumors result in strong lymphocyte suppression. Consequently, conventional immunotherapies targeting CD8 T cells are ineffective against brain tumors. Tumor cells escape immunosurveillance by various mechanisms and tumor cell metabolism can affect the metabolic states and functions of tumor-infiltrating lymphocytes. Here, we discovered that brain tumor cells had a particularly high demand for oxygen, which affected γδ T cell-mediated antitumor immune responses but not those of conventional T cells. Specifically, tumor hypoxia activated the γδ T cell protein kinase A pathway at a transcriptional level, resulting in repression of the activatory receptor NKG2D. Alleviating tumor hypoxia reinvigorated NKG2D expression and the antitumor function of γδ T cells. These results reveal a hypoxia-mediated mechanism through which brain tumors and γδ T cells interact and emphasize the importance of γδ T cells for antitumor immunity against brain tumors.
Assuntos
Neoplasias Encefálicas/imunologia , Citotoxicidade Imunológica , Glioblastoma/imunologia , Linfócitos Intraepiteliais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Evasão Tumoral , Microambiente Tumoral , Animais , Apoptose , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Antígenos CD8/genética , Antígenos CD8/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes Codificadores da Cadeia delta de Receptores de Linfócitos T , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Linfócitos Intraepiteliais/metabolismo , Linfócitos Intraepiteliais/patologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos Nus , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Fenótipo , Transdução de Sinais , Hipóxia TumoralRESUMO
Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.
Assuntos
Neoplasias da Mama/patologia , Heterogeneidade Genética , Organoides/patologia , Bancos de Tecidos , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Células Cultivadas , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Feminino , Humanos , Camundongos , Camundongos Nus , Organoides/efeitos dos fármacos , Medicina de Precisão/métodosRESUMO
Tandem repeats (TRs) are generated by DNA replication errors and retain a high level of instability, which in principle would make them unsuitable for integration into gene regulatory networks. However, the appearance of DNA sequence motifs recognized by transcription factors may turn TRs into functional cis-regulatory elements, thus favoring their stabilization in genomes. Here, we show that, in human cells, the transcriptional repressor ZEB1, which promotes the maintenance of mesenchymal features largely by suppressing epithelial genes and microRNAs, occupies TRs harboring dozens of copies of its DNA-binding motif within genomic loci relevant for maintenance of epithelial identity. The deletion of one such TR caused quasi-mesenchymal cancer cells to reacquire epithelial features, partially recapitulating the effects of ZEB1 gene deletion. These data demonstrate that the high density of identical motifs in TRs can make them suitable platforms for recruitment of transcriptional repressors, thus promoting their exaptation into pre-existing cis-regulatory networks.
Assuntos
Sequências de Repetição em Tandem/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Adulto , Animais , Sequência de Bases , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Feminino , Expressão Gênica , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Mucosa Bucal/metabolismo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Fatores de Transcrição/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/deficiência , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.