RESUMEN
Tissue damage increases the risk of cancer through poorly understood mechanisms1. In mouse models of pancreatic cancer, pancreatitis associated with tissue injury collaborates with activating mutations in the Kras oncogene to markedly accelerate the formation of early neoplastic lesions and, ultimately, adenocarcinoma2,3. Here, by integrating genomics, single-cell chromatin assays and spatiotemporally controlled functional perturbations in autochthonous mouse models, we show that the combination of Kras mutation and tissue damage promotes a unique chromatin state in the pancreatic epithelium that distinguishes neoplastic transformation from normal regeneration and is selected for throughout malignant evolution. This cancer-associated epigenetic state emerges within 48 hours of pancreatic injury, and involves an 'acinar-to-neoplasia' chromatin switch that contributes to the early dysregulation of genes that define human pancreatic cancer. Among the factors that are most rapidly activated after tissue damage in the pre-malignant pancreatic epithelium is the alarmin cytokine interleukin 33, which recapitulates the effects of injury in cooperating with mutant Kras to unleash the epigenetic remodelling program of early neoplasia and neoplastic transformation. Collectively, our study demonstrates how gene-environment interactions can rapidly produce gene-regulatory programs that dictate early neoplastic commitment, and provides a molecular framework for understanding the interplay between genetic and environmental cues in the initiation of cancer.
Asunto(s)
Transformación Celular Neoplásica/genética , Epigénesis Genética , Interacción Gen-Ambiente , Páncreas/metabolismo , Páncreas/patología , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Transformación Celular Neoplásica/patología , Cromatina/genética , Cromatina/metabolismo , Cromatina/patología , Modelos Animales de Enfermedad , Femenino , Genómica , Humanos , Interleucina-33/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
High-grade serous ovarian carcinoma (HGSOC) is a cancer with dismal prognosis due to the limited effectiveness of existing chemo- and immunotherapies. To elucidate mechanisms mediating sensitivity or resistance to these therapies, we developed a fast and flexible autochthonous mouse model based on somatic introduction of HGSOC-associated genetic alterations into the ovary of immunocompetent mice using tissue electroporation. Tumors arising in these mice recapitulate the metastatic patterns and histological, molecular, and treatment response features of the human disease. By leveraging these models, we show that the ability to undergo senescence underlies the clinically observed increase in sensitivity of homologous recombination (HR)-deficient HGSOC tumors to platinum-based chemotherapy. Further, cGas/STING-mediated activation of a restricted senescence-associated secretory phenotype (SASP) was sufficient to induce immune infiltration and sensitize HR-deficient tumors to immune checkpoint blockade. In sum, our study identifies senescence propensity as a predictor of therapy response and defines a limited SASP profile that appears sufficient to confer added vulnerability to concurrent immunotherapy and, more broadly, provides a blueprint for the implementation of electroporation-based mouse models to reveal mechanisms of oncogenesis and therapy response in HGSOC.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Puntos de Control Inmunológico/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Animales , Carcinoma Epitelial de Ovario/dietoterapia , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoterapia/métodos , Ratones , Ratones Endogámicos C57BLRESUMEN
The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3'UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3'UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A-eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5'-3' end interaction, mediated by both viral and cellular proteins.
Asunto(s)
Proteínas de la Nucleocápside/metabolismo , Orthohantavirus/genética , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas/fisiología , ARN Viral/genética , Proteínas de Unión al ARN/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Humanos , ARN Mensajero/genéticaRESUMEN
Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal co-activation of these pathways in the adult Drosophila midgut induces "tumor-like" overgrowths. Here, we show that, in these clones and in CRC cell lines, Dpp/TGF-ß acts as a tumor suppressor. Moreover, we discover that the Iroquois/IRX-family-protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. We also show that this genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF-ß. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF-ß. In addition, Irx5 expression confers a growth advantage in the presence of TGF-ß, but is selected against in its absence. Together, our results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF-ß activity. We propose that during the characteristic adenoma-to-carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF-ß, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF-ß pathway components.
Asunto(s)
Adenocarcinoma/metabolismo , Carcinogénesis/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Células Cultivadas , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/genética , Humanos , Mucosa Intestinal/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Regulación hacia ArribaRESUMEN
The elimination of large genomic regions has been enabled by the advent of site-specific nucleases. However, as the intended deletions get larger, the efficiency of successful engineering decreases to a point where it is not feasible to retrieve edited cells due to the rarity of on-target events. To address this issue, we developed a system called molecular alteration of chromosomes with engineered tandem elements (MACHETE). MACHETE is a CRISPR-Cas9-based system involving two stages: the initial insertion of a bicistronic positive/negative selection cassette to the locus of interest. This is followed by the introduction of single-guide RNAs flanking the knockin cassette to engineer the intended deletion, where only cells that have lost the locus survive the negative selection. In contrast to other approaches optimizing the activity of sequence-specific nucleases, MACHETE selects for the deletion event itself, thus greatly enriching for cells with the engineered alteration. The procedure routinely takes 4-6 weeks from design to selection of polyclonal populations bearing the deletion of interest. We have successfully deployed MACHETE to engineer deletions of up to 45 Mb, as well as the rapid creation of allelic series to map the relevant activities within a locus. This protocol details the design and step-by-step procedure to engineer megabase-sized deletions in cells of interest, with potential application for cancer genetics, transcriptional regulation, genome architecture and beyond.
Asunto(s)
Sistemas CRISPR-Cas , Humanos , Cromosomas/genética , Edición Génica/métodos , Ingeniería Genética/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Eliminación de SecuenciaRESUMEN
Driver gene mutations can increase the metastatic potential of the primary tumor1-3, but their role in sustaining tumor growth at metastatic sites is poorly understood. A paradigm of such mutations is inactivation of SMAD4 - a transcriptional effector of TGFß signaling - which is a hallmark of multiple gastrointestinal malignancies4,5. SMAD4 inactivation mediates TGFß's remarkable anti- to pro-tumorigenic switch during cancer progression and can thus influence both tumor initiation and metastasis6-14. To determine whether metastatic tumors remain dependent on SMAD4 inactivation, we developed a mouse model of pancreatic ductal adenocarcinoma (PDAC) that enables Smad4 depletion in the pre-malignant pancreas and subsequent Smad4 reactivation in established metastases. As expected, Smad4 inactivation facilitated the formation of primary tumors that eventually colonized the liver and lungs. By contrast, Smad4 reactivation in metastatic disease had strikingly opposite effects depending on the tumor's organ of residence: suppression of liver metastases and promotion of lung metastases. Integrative multiomic analysis revealed organ-specific differences in the tumor cells' epigenomic state, whereby the liver and lungs harbored chromatin programs respectively dominated by the KLF and RUNX developmental transcription factors, with Klf4 depletion being sufficient to reverse Smad4's tumor-suppressive activity in liver metastases. Our results show how epigenetic states favored by the organ of residence can influence the function of driver genes in metastatic tumors. This organ-specific gene-chromatin interplay invites consideration of anatomical site in the interpretation of tumor genetics, with implications for the therapeutic targeting of metastatic disease.
RESUMEN
Neural stem cells (NSCs) in the adult murine subependymal zone balance their self-renewal capacity and glial identity with the potential to generate neurons during the lifetime. Adult NSCs exhibit lineage priming via pro-neurogenic fate determinants. However, the protein levels of the neural fate determinants are not sufficient to drive direct differentiation of adult NSCs, which raises the question of how cells along the neurogenic lineage avoid different conflicting fate choices, such as self-renewal and differentiation. Here, we identify RNA-binding protein MEX3A as a post-transcriptional regulator of a set of stemness associated transcripts at critical transitions in the subependymal neurogenic lineage. MEX3A regulates a quiescence-related RNA signature in activated NSCs that is needed for their return to quiescence, playing a role in the long-term maintenance of the NSC pool. Furthermore, it is required for the repression of the same program at the onset of neuronal differentiation. Our data indicate that MEX3A is a pivotal regulator of adult murine neurogenesis acting as a translational remodeller.
Asunto(s)
Células-Madre Neurales , Neurogénesis , Ratones , Animales , Neurogénesis/genética , Neuronas/fisiología , Células-Madre Neurales/metabolismo , Diferenciación Celular/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The response to tumor-initiating inflammatory and genetic insults can vary among morphologically indistinguishable cells, suggesting as yet uncharacterized roles for epigenetic plasticity during early neoplasia. To investigate the origins and impact of such plasticity, we performed single-cell analyses on normal, inflamed, premalignant, and malignant tissues in autochthonous models of pancreatic cancer. We reproducibly identified heterogeneous cell states that are primed for diverse, late-emerging neoplastic fates and linked these to chromatin remodeling at cell-cell communication loci. Using an inference approach, we revealed signaling gene modules and tissue-level cross-talk, including a neoplasia-driving feedback loop between discrete epithelial and immune cell populations that was functionally validated in mice. Our results uncover a neoplasia-specific tissue-remodeling program that may be exploited for pancreatic cancer interception.
Asunto(s)
Carcinogénesis , Epigénesis Genética , Páncreas , Neoplasias Pancreáticas , Animales , Ratones , Carcinogénesis/genética , Carcinogénesis/patología , Comunicación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Páncreas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologíaRESUMEN
KRAS mutation in colorectal cancer is associated with aggressive tumor behavior through increased invasiveness and higher rates of lung metastases, but the biological mechanisms behind these features are not fully understood. In this study, we show that KRAS-mutant colorectal cancer upregulates integrin α6ß4 through ERK/MEK signaling. Knocking-out integrin ß4 (ITGB4) specifically depleted the expression of integrin α6ß4 and this resulted in a reduction in the invasion and migration ability of the cancer cells. We also observed a reduction in the number and area of lung metastatic foci in mice that were injected with ITGB4 knockout KRAS-mutant colorectal cancer cells compared with the mice injected with ITGB4 wild-type KRAS-mutant colorectal cancer cells, while no difference was observed in liver metastases. Inhibiting integrin α6ß4 in KRAS-mutant colorectal cancer could be a potential therapeutic target to diminish the KRAS-invasive phenotype and associated pulmonary metastasis rate. IMPLICATIONS: Knocking-out ITGB4, which is overexpressed in KRAS-mutant colorectal cancer and promotes tumor aggressiveness, diminishes local invasiveness and rates of pulmonary metastasis.
Asunto(s)
Neoplasias Colorrectales , Integrina beta4 , Neoplasias Pulmonares , Animales , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Integrina beta4/genética , Integrina beta4/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Ratones , Invasividad Neoplásica/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
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.
Asunto(s)
Interferones , Neoplasias , Animales , Ratones , Deleción Cromosómica , Cromosomas , Evasión Inmune , Microambiente Tumoral/genética , Secuencias Repetidas en TándemRESUMEN
We describe a mouse model of rectal cancer (RC) involving rapid tumor organoid engraftment via orthotopic transplantation in an immunocompetent setting. This approach uses simple mechanical disruption to allow engraftment, avoiding the use of dextran sulfate sodium. The resulting RC tumors invaded from the mucosal surface and metastasized to distant organs. Histologically, the tumors closely resemble human RC and mirror remodeling of the tumor microenvironment in response to radiation. This murine RC model thus recapitulates key aspects of human RC pathogenesis and presents an accessible approach for more physiologically accurate, preclinical efficacy studies.
Asunto(s)
Neoplasias del Recto , Ratones , Humanos , Animales , Neoplasias del Recto/radioterapia , Microambiente TumoralRESUMEN
Colorectal cancer (CRC) patient-derived organoids predict responses to chemotherapy. Here we used them to investigate relapse after treatment. Patient-derived organoids expand from highly proliferative LGR5+ tumor cells; however, we discovered that lack of optimal growth conditions specifies a latent LGR5+ cell state. This cell population expressed the gene MEX3A, is chemoresistant and regenerated the organoid culture after treatment. In CRC mouse models, Mex3a+ cells contributed marginally to metastatic outgrowth; however, after chemotherapy, Mex3a+ cells produced large cell clones that regenerated the disease. Lineage-tracing analysis showed that persister Mex3a+ cells downregulate the WNT/stem cell gene program immediately after chemotherapy and adopt a transient state reminiscent to that of YAP+ fetal intestinal progenitors. In contrast, Mex3a-deficient cells differentiated toward a goblet cell-like phenotype and were unable to resist chemotherapy. Our findings reveal that adaptation of cancer stem cells to suboptimal niche environments protects them from chemotherapy and identify a candidate cell of origin of relapse after treatment in CRC.
Asunto(s)
Neoplasias Colorrectales , Organoides , Animales , Diferenciación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Ratones , Células Madre Neoplásicas , RecurrenciaRESUMEN
Base editing can be applied to characterize single nucleotide variants of unknown function, yet defining effective combinations of single guide RNAs (sgRNAs) and base editors remains challenging. Here, we describe modular base-editing-activity 'sensors' that link sgRNAs and cognate target sites in cis and use them to systematically measure the editing efficiency and precision of thousands of sgRNAs paired with functionally distinct base editors. By quantifying sensor editing across >200,000 editor-sgRNA combinations, we provide a comprehensive resource of sgRNAs for introducing and interrogating cancer-associated single nucleotide variants in multiple model systems. We demonstrate that sensor-validated tools streamline production of in vivo cancer models and that integrating sensor modules in pooled sgRNA libraries can aid interpretation of high-throughput base editing screens. Using this approach, we identify several previously uncharacterized mutant TP53 alleles as drivers of cancer cell proliferation and in vivo tumor development. We anticipate that the framework described here will facilitate the functional interrogation of cancer variants in cell and animal models.
Asunto(s)
Edición Génica , Neoplasias , Animales , Sistemas CRISPR-Cas/genética , Neoplasias/genética , Nucleótidos , ARN Guía de Kinetoplastida/genéticaRESUMEN
Somatic mutations in the KRAS oncogene are associated with poor outcomes in locally advanced rectal cancer but the underlying biologic mechanisms are not fully understood. We profiled mRNA in 76 locally advanced rectal adenocarcinomas from patients that were enrolled in a prospective clinical trial and investigated differences in gene expression between KRAS mutant (KRAS-mt) and KRAS-wild-type (KRAS-wt) patients. We found that KRAS-mt tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix. We validated our findings using samples from The Cancer Genome Atlas (TCGA) and also by performing immunohistochemistry (IHC) and immunofluorescence (IF) in orthogonal cohorts. Using in vitro and in vivo models, we show that oncogenic KRAS signaling within the epithelial cancer cells modulates the activity of the surrounding fibroblasts in the tumor microenvironment.
Asunto(s)
Proteínas Proto-Oncogénicas p21(ras) , Neoplasias del Recto , Ensayos Clínicos como Asunto , Matriz Extracelular , Fibroblastos/patología , Humanos , Mutación/genética , Estudios Prospectivos , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias del Recto/genética , Neoplasias del Recto/patología , Microambiente TumoralRESUMEN
To study genetic factors influencing the progression and therapeutic responses of advanced prostate cancer, we developed a fast and flexible system that introduces genetic alterations relevant to human disease directly into the prostate glands of mice using tissue electroporation. These electroporation-based genetically engineered mouse models (EPO-GEMM) recapitulate features of traditional germline models and, by modeling genetic factors linked to late-stage human disease, can produce tumors that are metastatic and castration-resistant. A subset of tumors with Trp53 alterations acquired spontaneous WNT pathway alterations, which are also associated with metastatic prostate cancer in humans. Using the EPO-GEMM approach and an orthogonal organoid-based model, we show that WNT pathway activation drives metastatic disease that is sensitive to pharmacologic WNT pathway inhibition. Thus, by leveraging EPO-GEMMs, we reveal a functional role for WNT signaling in driving prostate cancer metastasis and validate the WNT pathway as therapeutic target in metastatic prostate cancer. SIGNIFICANCE: Our understanding of the factors driving metastatic prostate cancer is limited by the paucity of models of late-stage disease. Here, we develop EPO-GEMMs of prostate cancer and use them to identify and validate the WNT pathway as an actionable driver of aggressive metastatic disease.This article is highlighted in the In This Issue feature, p. 890.
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Neoplasias de la Próstata/genética , Ingeniería de Tejidos/métodos , Vía de Señalización Wnt/genética , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Metástasis de la NeoplasiaRESUMEN
Intestinal organoids have emerged as a powerful in vitro tool for studying intestinal biology due to their resemblance to in vivo tissue at the structural and functional levels. However, their sphere-like geometry prevents access to the apical side of the epithelium, making them unsuitable for standard functional assays designed for flat cell monolayers. Here, we describe a simple method for the formation of epithelial monolayers that recapitulates the in vivo-like cell type composition and organization and that is suitable for functional tissue barrier assays. In our approach, epithelial monolayer spreading is driven by the substrate stiffness, while tissue barrier function is achieved by the basolateral delivery of medium enriched with stem cell niche and myofibroblast-derived factors. These monolayers contain major intestinal epithelial cell types organized into proliferating crypt-like domains and differentiated villus-like regions, closely resembling the in vivo cell distribution. As a unique characteristic, these epithelial monolayers form functional epithelial barriers with an accessible apical surface and physiologically relevant transepithelial electrical resistance values. Our technology offers an up-to-date and novel culture method for intestinal epithelium, providing an in vivo-like cell composition and distribution in a tissue culture format compatible with high-throughput drug absorption or microbe-epithelium interaction studies.
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Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Intestino Delgado/citología , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Colágeno , Medios de Cultivo Condicionados/farmacología , Combinación de Medicamentos , Proteínas Fluorescentes Verdes/genética , Laminina , Membranas Artificiales , Organoides , Proteoglicanos , Proteína Wnt3A/metabolismoRESUMEN
Metastasis underlies the majority of cancer-related deaths yet remains poorly understood due, in part, to the lack of models in vivo. Here we show that expression of the EMT master inducer Snail in primary adult Drosophila intestinal tumors leads to the dissemination of tumor cells and formation of macrometastases. Snail drives an EMT in tumor cells, which, although retaining some epithelial markers, subsequently break through the basal lamina of the midgut, undergo a collective migration and seed polyclonal metastases. While metastases re-epithelialize over time, we found that early metastases are remarkably mesenchymal, discarding the requirement for a mesenchymal-to-epithelial transition for early stages of metastatic growth. Our results demonstrate the formation of metastases in adult flies, and identify a key role for partial-EMTs in driving it. This model opens the door to investigate the basic mechanisms underlying metastasis, in a powerful in vivo system suited for rapid genetic and drug screens.
Asunto(s)
Movimiento Celular , Transición Epitelial-Mesenquimal , Neoplasias Intestinales/patología , Factores de Transcripción de la Familia Snail/metabolismo , Animales , Drosophila melanogaster , Femenino , Neoplasias Experimentales/patologíaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Highly proliferative Lgr5+ stem cells maintain the intestinal epithelium and are thought to be largely homogeneous. Although quiescent intestinal stem cell (ISC) populations have been described, the identity and features of such a population remain controversial. Here we report unanticipated heterogeneity within the Lgr5+ ISC pool. We found that expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages with distinct kinetics. Single-cell transcriptome profiling revealed that Lgr5+ cells adopt two discrete states, one of which is defined by a Mex3a expression program and relatively low levels of proliferation genes. During homeostasis, Mex3a+ cells continually shift into the rapidly dividing, self-renewing ISC pool. Chemotherapy and radiation preferentially target rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, helping to promote regeneration of the intestinal epithelium following toxic insults. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment.