RESUMEN
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.
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Anticuerpos/uso terapéutico , Aprendizaje Profundo , Diagnóstico por Computador/métodos , Quimioterapia Asistida por Computador/métodos , Neoplasias/patología , Animales , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Ratones SCID , Metástasis de la Neoplasia , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Programas Informáticos , Microambiente TumoralRESUMEN
Memories about sensory experiences are tightly linked to the context in which they were formed. Memory contextualization is fundamental for the selection of appropriate behavioral reactions needed for survival, yet the underlying neuronal circuits are poorly understood. By combining trans-synaptic viral tracing and optogenetic manipulation, we found that the ventral hippocampus (vHC) and the amygdala, two key brain structures encoding context and emotional experiences, interact via multiple parallel pathways. A projection from the vHC to the basal amygdala mediates fear behavior elicited by a conditioned context, whereas a parallel projection from a distinct subset of vHC neurons onto midbrain-projecting neurons in the central amygdala is necessary for context-dependent retrieval of cued fear memories. Our findings demonstrate that two fundamentally distinct roles of context in fear memory retrieval are processed by distinct vHC output pathways, thereby allowing for the formation of robust contextual fear memories while preserving context-dependent behavioral flexibility.
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Amígdala del Cerebelo/fisiología , Hipocampo/fisiología , Memoria , Vías Nerviosas , Animales , Condicionamiento Psicológico , Fenómenos Electrofisiológicos , Miedo , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/fisiología , Optogenética , Virus de la Rabia/genética , SinapsisRESUMEN
KRAS is one of the most frequently mutated oncogenes in human cancer. Despite substantial efforts, no clinically applicable strategy has yet been developed to effectively treat KRAS-mutant tumors. Here, we perform a cell-line-based screen and identify strong synergistic interactions between cell-cycle checkpoint-abrogating Chk1- and MK2 inhibitors, specifically in KRAS- and BRAF-driven cells. Mechanistically, we show that KRAS-mutant cancer displays intrinsic genotoxic stress, leading to tonic Chk1- and MK2 activity. We demonstrate that simultaneous Chk1- and MK2 inhibition leads to mitotic catastrophe in KRAS-mutant cells. This actionable synergistic interaction is validated using xenograft models, as well as distinct Kras- or Braf-driven autochthonous murine cancer models. Lastly, we show that combined checkpoint inhibition induces apoptotic cell death in KRAS- or BRAF-mutant tumor cells directly isolated from patients. These results strongly recommend simultaneous Chk1- and MK2 inhibition as a therapeutic strategy for the treatment of KRAS- or BRAF-driven cancers.
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Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas ras/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma del Pulmón , Animales , Puntos de Control del Ciclo Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Modelos Animales de Enfermedad , Xenoinjertos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Ratones , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras) , Células Tumorales CultivadasRESUMEN
Although long noncoding RNAs (lncRNAs) dominate the transcriptome, their functions are largely unexplored. The extensive overlap of lncRNAs with coding and regulatory sequences restricts their systematic interrogation by DNA-directed perturbation. Here we developed genome-scale lncRNA transcriptome screening using Cas13d/CasRx. We show that RNA targeting overcomes limitations inherent to other screening methods, thereby considerably expanding the explorable space of the lncRNAome. By evolving the screening system toward pan-cancer applicability, it supports molecular and phenotypic data integration to contextualize screening hits or infer lncRNA function. We thereby addressed challenges posed by the enormous transcriptome size and tissue specificity through a size-reduced multiplexed gRNA library termed Albarossa, targeting 24,171 lncRNA genes. Its rational design incorporates target prioritization based on expression, evolutionary conservation and tissue specificity, thereby reconciling high discovery power and pan-cancer representation with scalable experimental throughput. Applied across entities, the screening platform identified numerous context-specific and common essential lncRNAs. Our work sets the stage for systematic exploration of lncRNA biology in health and disease.
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Neoplasias , ARN Largo no Codificante , Humanos , Perfilación de la Expresión Génica , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Guía de Sistemas CRISPR-Cas , Transcriptoma , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
The EMT-transcription factor ZEB1 is heterogeneously expressed in tumor cells and in cancer-associated fibroblasts (CAFs) in colorectal cancer (CRC). While ZEB1 in tumor cells regulates metastasis and therapy resistance, its role in CAFs is largely unknown. Combining fibroblast-specific Zeb1 deletion with immunocompetent mouse models of CRC, we observe that inflammation-driven tumorigenesis is accelerated, whereas invasion and metastasis in sporadic cancers are reduced. Single-cell transcriptomics, histological characterization, and in vitro modeling reveal a crucial role of ZEB1 in CAF polarization, promoting myofibroblastic features by restricting inflammatory activation. Zeb1 deficiency impairs collagen deposition and CAF barrier function but increases NFκB-mediated cytokine production, jointly promoting lymphocyte recruitment and immune checkpoint activation. Strikingly, the Zeb1-deficient CAF repertoire sensitizes to immune checkpoint inhibition, offering a therapeutic opportunity of targeting ZEB1 in CAFs and its usage as a prognostic biomarker. Collectively, we demonstrate that ZEB1-dependent plasticity of CAFs suppresses anti-tumor immunity and promotes metastasis.
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Fibroblastos Asociados al Cáncer , Neoplasias Colorrectales , Inmunoterapia , Inflamación , Homeobox 1 de Unión a la E-Box con Dedos de Zinc , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/terapia , Neoplasias Colorrectales/inmunología , Animales , Ratones , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Humanos , Inflamación/metabolismo , Inflamación/genética , Inflamación/patología , Inmunoterapia/métodos , Regulación Neoplásica de la Expresión Génica , Fibroblastos/metabolismo , Línea Celular Tumoral , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Transición Epitelial-Mesenquimal/genéticaRESUMEN
The laboratory mouse ranks among the most important experimental systems for biomedical research and molecular reference maps of such models are essential informational tools. Here, we present a quantitative draft of the mouse proteome and phosphoproteome constructed from 41 healthy tissues and several lines of analyses exemplify which insights can be gleaned from the data. For instance, tissue- and cell-type resolved profiles provide protein evidence for the expression of 17,000 genes, thousands of isoforms and 50,000 phosphorylation sites in vivo. Proteogenomic comparison of mouse, human and Arabidopsis reveal common and distinct mechanisms of gene expression regulation and, despite many similarities, numerous differentially abundant orthologs that likely serve species-specific functions. We leverage the mouse proteome by integrating phenotypic drug (n > 400) and radiation response data with the proteomes of 66 pancreatic ductal adenocarcinoma (PDAC) cell lines to reveal molecular markers for sensitivity and resistance. This unique atlas complements other molecular resources for the mouse and can be explored online via ProteomicsDB and PACiFIC.
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Arabidopsis , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Arabidopsis/genética , Carcinoma Ductal Pancreático/metabolismo , Espectrometría de Masas , Ratones , Neoplasias Pancreáticas/genética , Proteoma/análisisRESUMEN
OBJECTIVE: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC). DESIGN: To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumours in mice. RESULTS: Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immunoevasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer. CONCLUSION: One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.
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ATPasas Asociadas con Actividades Celulares Diversas , Carcinoma Ductal Pancreático , ADN Helicasas , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas c-myc , Animales , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Ratones , Humanos , ADN Helicasas/genética , ADN Helicasas/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Línea Celular Tumoral , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genéticaRESUMEN
Surgical resection, when combined with chemotherapy, has been shown to significantly improve the survival rate of patients with pancreatic ductal adenocarcinoma (PDAC). However, this treatment option is only feasible for a fraction of patients, as more than 50% of cases are diagnosed with metastasis. The multifaceted process of metastasis is still not fully understood, but recent data suggest that transcriptional and epigenetic plasticity play significant roles. Interfering with epigenetic reprogramming can potentially control the adaptive processes responsible for metastatic progression and therapy resistance, thereby enhancing treatment responses and preventing recurrence. This review will focus on the relevance of histone-modifying enzymes in pancreatic cancer, specifically on their impact on the metastatic cascade. Additionally, it will also provide a brief update on the current clinical developments in epigenetic therapies.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Epigénesis GenéticaRESUMEN
Epithelial differentiation of normal and tumor cells is orchestrated by lineage-determining transcriptional regulatory networks that enforce cell identity. Recent research by Kalisz et al (2020) in the EMBO Journal elucidates the molecular mechanisms by which a transcriptional differentiation program governed by HNF1A and KDM6A maintains acinar differentiation and the epithelial identity of pancreatic ductal adenocarcinoma (PDAC). Loss of function of either transcriptional regulator induces tumor progression to a poorly differentiated and highly aggressive PDAC subtype with a squamous transcriptome and poor prognosis.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Células Acinares , Diferenciación Celular , Factor Nuclear 1-alfa del Hepatocito , Histona Demetilasas , HumanosRESUMEN
MOTIVATION: Cancer is one of the leading causes of death worldwide. Despite significant improvements in prevention and treatment, mortality remains high for many cancer types. Hence, innovative methods that use molecular data to stratify patients and identify biomarkers are needed. Promising biomarkers can also be inferred from competing endogenous RNA (ceRNA) networks that capture the gene-miRNA gene regulatory landscape. Thus far, the role of these biomarkers could only be studied globally but not in a sample-specific manner. To mitigate this, we introduce spongEffects, a novel method that infers subnetworks (or modules) from ceRNA networks and calculates patient- or sample-specific scores related to their regulatory activity. RESULTS: We show how spongEffects can be used for downstream interpretation and machine learning tasks such as tumor classification and for identifying subtype-specific regulatory interactions. In a concrete example of breast cancer subtype classification, we prioritize modules impacting the biology of the different subtypes. In summary, spongEffects prioritizes ceRNA modules as biomarkers and offers insights into the miRNA regulatory landscape. Notably, these module scores can be inferred from gene expression data alone and can thus be applied to cohorts where miRNA expression information is lacking. AVAILABILITY AND IMPLEMENTATION: https://bioconductor.org/packages/devel/bioc/html/SPONGE.html.
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Neoplasias de la Mama , MicroARNs , ARN Largo no Codificante , Humanos , Femenino , MicroARNs/genética , MicroARNs/metabolismo , Redes Reguladoras de Genes , Neoplasias de la Mama/genética , Aprendizaje Automático , Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante/genéticaRESUMEN
The poor correlation of mutational landscapes with phenotypes limits our understanding of the pathogenesis and metastasis of pancreatic ductal adenocarcinoma (PDAC). Here we show that oncogenic dosage-variation has a critical role in PDAC biology and phenotypic diversification. We find an increase in gene dosage of mutant KRAS in human PDAC precursors, which drives both early tumorigenesis and metastasis and thus rationalizes early PDAC dissemination. To overcome the limitations posed to gene dosage studies by the stromal richness of PDAC, we have developed large cell culture resources of metastatic mouse PDAC. Integration of cell culture genomes, transcriptomes and tumour phenotypes with functional studies and human data reveals additional widespread effects of oncogenic dosage variation on cell morphology and plasticity, histopathology and clinical outcome, with the highest KrasMUT levels underlying aggressive undifferentiated phenotypes. We also identify alternative oncogenic gains (Myc, Yap1 or Nfkb2), which collaborate with heterozygous KrasMUT in driving tumorigenesis, but have lower metastatic potential. Mechanistically, different oncogenic gains and dosages evolve along distinct evolutionary routes, licensed by defined allelic states and/or combinations of hallmark tumour suppressor alterations (Cdkn2a, Trp53, Tgfß-pathway). Thus, evolutionary constraints and contingencies direct oncogenic dosage gain and variation along defined routes to drive the early progression of PDAC and shape its downstream biology. Our study uncovers universal principles of Ras-driven oncogenesis that have potential relevance beyond pancreatic cancer.
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Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Evolución Molecular , Dosificación de Gen , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Alelos , Animales , Carcinogénesis/genética , Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Progresión de la Enfermedad , Femenino , Genes myc , Genes p53 , Humanos , Masculino , Ratones , Mutación , Subunidad p52 de NF-kappa B/genética , Metástasis de la Neoplasia/genética , Proteínas Nucleares/genética , Fenotipo , Fosfoproteínas/genética , Factores de Transcripción/genética , Transcriptoma/genética , Factor de Crecimiento Transformador beta1/genética , Proteínas Señalizadoras YAPRESUMEN
Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 µm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.
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Detección Precoz del Cáncer/métodos , Endoscopía/métodos , Lesiones Precancerosas/diagnóstico , Animales , Colon/patología , Neoplasias del Colon/patología , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Femenino , Fluorescencia , Colorantes Fluorescentes , Neoplasias Gastrointestinales/patología , Tracto Gastrointestinal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Imagen Molecular/métodos , Lesiones Precancerosas/patología , Ratas , Ratas Endogámicas , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/prevención & control , PorcinosRESUMEN
BACKGROUND & AIMS: The Notch signaling pathway is an important pathway in the adult pancreas and in pancreatic ductal adenocarcinoma (PDAC), with hairy and enhancer of split-1 (HES1) as the core molecule in this pathway. However, the roles of HES1 in the adult pancreas and PDAC formation remain controversial. METHODS: We used genetically engineered dual-recombinase mouse models for inducing Hes1 deletion under various conditions. RESULTS: The loss of Hes1 expression in the adult pancreas did not induce phenotypic alterations. However, regeneration was impaired after caerulein-induced acute pancreatitis. In a pancreatic intraepithelial neoplasia (PanIN) mouse model, PanINs rarely formed when Hes1 deletion preceded PanIN formation, whereas more PanINs were formed when Hes1 deletion succeeded PanIN formation. In a PDAC mouse model, PDAC formation was also enhanced by Hes1 deletion after PanIN/PDAC development; therefore, Hes1 promotes PanIN initiation but inhibits PanIN/PDAC progression. RNA sequencing and chromatin immunoprecipitation-quantitative polymerase chain reaction revealed that Hes1 deletion enhanced epithelial-to-mesenchymal transition via Muc5ac up-regulation in PDAC progression. The results indicated that HES1 is not required for maintaining the adult pancreas under normal conditions, but is important for regeneration during recovery from pancreatitis; moreover, Hes1 plays different roles, depending on the tumor condition. CONCLUSIONS: Our findings highlight the context-dependent roles of HES1 in the adult pancreas and pancreatic cancer.
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Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Pancreatitis , Animales , Ratones , Enfermedad Aguda , Pancreatitis/inducido químicamente , Pancreatitis/genética , Páncreas , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/genética , Factor de Transcripción HES-1/genética , Neoplasias PancreáticasRESUMEN
BACKGROUND & AIMS: Throughout life, the intestinal epithelium undergoes constant self-renewal from intestinal stem cells. Together with genotoxic stressors and failing DNA repair, this self-renewal causes susceptibility toward malignant transformation. X-box binding protein 1 (XBP1) is a stress sensor involved in the unfolded protein response (UPR). We hypothesized that XBP1 acts as a signaling hub to regulate epithelial DNA damage responses. METHODS: Data from The Cancer Genome Atlas were analyzed for association of XBP1 with colorectal cancer (CRC) survival and molecular interactions between XBP1 and p53 pathway activity. The role of XBP1 in orchestrating p53-driven DNA damage response was tested in vitro in mouse models of chronic intestinal epithelial cell (IEC) DNA damage (Xbp1/H2bfl/fl, Xbp1ΔIEC, H2bΔIEC, H2b/Xbp1ΔIEC) and via orthotopic tumor organoid transplantation. Transcriptome analysis of intestinal organoids was performed to identify molecular targets of Xbp1-mediated DNA damage response. RESULTS: In The Cancer Genome Atlas data set of CRC, low XBP1 expression was significantly associated with poor overall survival and reduced p53 pathway activity. In vivo, H2b/Xbp1ΔIEC mice developed spontaneous intestinal carcinomas. Orthotopic tumor organoid transplantation revealed a metastatic potential of H2b/Xbp1ΔIEC-derived tumors. RNA sequencing of intestinal organoids (H2b/Xbp1fl/fl, H2bΔIEC, H2b/Xbp1ΔIEC, and H2b/p53ΔIEC) identified a transcriptional program downstream of p53, in which XBP1 directs DNA-damage-inducible transcript 4-like (Ddit4l) expression. DDIT4L inhibits mechanistic target of rapamycin-mediated phosphorylation of 4E-binding protein 1. Pharmacologic mechanistic target of rapamycin inhibition suppressed epithelial hyperproliferation via 4E-binding protein 1. CONCLUSIONS: Our data suggest a crucial role for XBP1 in coordinating epithelial DNA damage responses and stem cell function via a p53-DDIT4L-dependent feedback mechanism.
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Adenocarcinoma/metabolismo , Adenoma/metabolismo , Transformación Celular Neoplásica/metabolismo , Daño del ADN , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Neoplasias Intestinales/metabolismo , Proteína 1 de Unión a la X-Box/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenoma/tratamiento farmacológico , Adenoma/genética , Adenoma/patología , Animales , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Bases de Datos Genéticas , Estrés del Retículo Endoplásmico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Neoplasias Intestinales/tratamiento farmacológico , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Inhibidores mTOR/farmacología , Ratones Noqueados , Transducción de Señal , Sirolimus/farmacología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión a la X-Box/genéticaRESUMEN
BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma that contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown. METHODS: We used a dual recombinase mouse model to delete Hif1α or Hif2α in α-smooth muscle actin-expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF HIF2α expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, reverse transcription quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, an HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade. RESULTS: CAF-specific deletion of Hif2α, but not Hif1α, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n = 21-23 mice/group, Log-rank P = .0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models. CONCLUSIONS: Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.
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Fibroblastos Asociados al Cáncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma Ductal Pancreático/patología , Humanos , Hipoxia/metabolismo , Inhibidores de Puntos de Control Inmunológico , Terapia de Inmunosupresión , Ratones , Neoplasias Pancreáticas/patología , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Targeting KRAS downstream signaling remains an important therapeutic approach in pancreatic cancer. We used primary pancreatic ductal epithelial cells and mouse models allowing the conditional expression of oncogenic KrasG12D, to investigate KRAS signaling integrators. We observed that the AP1 family member FRA1 is tightly linked to the KRAS signal and expressed in pre-malignant lesions and the basal-like subtype of pancreatic cancer. However, genetic-loss-of-function experiments revealed that FRA1 is dispensable for KrasG12D-induced pancreatic cancer development in mice. Using FRA1 gain- and loss-of-function models in an unbiased drug screen, we observed that FRA1 is a modulator of the responsiveness of pancreatic cancer to inhibitors of the RAF-MEK-ERK cascade. Mechanistically, context-dependent FRA1-associated adaptive rewiring of oncogenic ERK signaling was observed and correlated with sensitivity to inhibitors of canonical KRAS signaling. Furthermore, pharmacological-induced degradation of FRA1 synergizes with MEK inhibitors. Our studies establish FRA1 as a part of the molecular machinery controlling sensitivity to MAPK cascade inhibition allowing the development of mechanism-based therapies.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas c-fos , Animales , Ratones , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Mutación , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras) , Proteínas Proto-Oncogénicas c-fos/metabolismo , Neoplasias PancreáticasRESUMEN
OBJECTIVE: Molecular taxonomy of tumours is the foundation of personalised medicine and is becoming of paramount importance for therapeutic purposes. Four transcriptomics-based classification systems of pancreatic ductal adenocarcinoma (PDAC) exist, which consistently identified a subtype of highly aggressive PDACs with basal-like features, including ΔNp63 expression and loss of the epithelial master regulator GATA6. We investigated the precise molecular events driving PDAC progression and the emergence of the basal programme. DESIGN: We combined the analysis of patient-derived transcriptomics datasets and tissue samples with mechanistic experiments using a novel dual-recombinase mouse model for Gata6 deletion at late stages of KRasG12D-driven pancreatic tumorigenesis (Gata6LateKO). RESULTS: This comprehensive human-to-mouse approach showed that GATA6 loss is necessary, but not sufficient, for the expression of ΔNp63 and the basal programme in patients and in mice. The concomitant loss of HNF1A and HNF4A, likely through epigenetic silencing, is required for the full phenotype switch. Moreover, Gata6 deletion in mice dramatically increased the metastatic rate, with a propensity for lung metastases. Through RNA-Seq analysis of primary cells isolated from mouse tumours, we show that Gata6 inhibits tumour cell plasticity and immune evasion, consistent with patient-derived data, suggesting that GATA6 works as a barrier for acquiring the fully developed basal and metastatic phenotype. CONCLUSIONS: Our work provides both a mechanistic molecular link between the basal phenotype and metastasis and a valuable preclinical tool to investigate the most aggressive subtype of PDAC. These data, therefore, are important for understanding the pathobiological features underlying the heterogeneity of pancreatic cancer in both mice and human.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Carcinoma Ductal Pancreático/patología , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Ratones , Neoplasias Pancreáticas/patología , Neoplasias PancreáticasRESUMEN
BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibroblast-rich desmoplastic stroma. Cancer-associated fibroblasts (CAFs) have been shown to display a high degree of interconvertible states including quiescent, inflammatory, and myofibroblastic phenotypes; however, the mechanisms by which this plasticity is achieved are poorly understood. Here, we aim to elucidate the role of CAF plasticity and its impact on PDAC biology. METHODS: To investigate the role of mesenchymal plasticity in PDAC progression, we generated a PDAC mouse model in which CAF plasticity is modulated by genetic depletion of the transcription factor Prrx1. Primary pancreatic fibroblasts from this mouse model were further characterized by functional in vitro assays. To characterize the impact of CAFs on tumor differentiation and response to chemotherapy, various coculture experiments were performed. In vivo, tumors were characterized by morphology, extracellular matrix composition, and tumor dissemination and metastasis. RESULTS: Our in vivo findings showed that Prrx1-deficient CAFs remain constitutively activated. Importantly, this CAF phenotype determines tumor differentiation and disrupts systemic tumor dissemination. Mechanistically, coculture experiments of tumor organoids and CAFs showed that CAFs shape the epithelial-to-mesenchymal phenotype and confer gemcitabine resistance of PDAC cells induced by CAF-derived hepatocyte growth factor. Furthermore, gene expression analysis showed that patients with pancreatic cancer with high stromal expression of Prrx1 display the squamous, most aggressive, subtype of PDAC. CONCLUSIONS: Here, we define that the Prrx1 transcription factor is critical for tuning CAF activation, allowing a dynamic switch between a dormant and an activated state. This work shows that Prrx1-mediated CAF plasticity has significant impact on PDAC biology and therapeutic resistance.
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Fibroblastos Asociados al Cáncer/fisiología , Carcinoma Ductal Pancreático/etiología , Carcinoma Ductal Pancreático/patología , Proteínas de Homeodominio/fisiología , Neoplasias Pancreáticas/etiología , Neoplasias Pancreáticas/patología , Animales , Plasticidad de la Célula/fisiología , Modelos Animales de Enfermedad , RatonesRESUMEN
Targeted protein degradation offers new opportunities to inactivate cancer drivers and has successfully entered the clinic. Ways to induce selective protein degradation include proteolysis targeting chimera (PROTAC) technology and immunomodulatory (IMiDs) / next-generation Cereblon (CRBN) E3 ligase modulating drugs (CELMoDs). Here, we aimed to develop a MYC PROTAC based on the MYC-MAX dimerization inhibitor 10058-F4 derivative 28RH and Thalidomide, called MDEG-541. We show that a subgroup of gastrointestinal cancer cell lines and primary patient-derived organoids are MDEG-541 sensitive. Although MYC expression was regulated in a CRBN-, proteasome- and ubiquitin-dependent manner, we provide evidence that MDEG-541 induced the degradation of CRBN neosubstrates, including G1 to S phase transition 1/2 (GSPT1/2) and the Polo-like kinase 1 (PLK1). In sum, we have established a CRBN-dependent degrader of relevant cancer targets with activity in gastrointestinal cancers.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Gastrointestinales/tratamiento farmacológico , Talidomida/farmacología , Tiazoles/farmacología , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Gastrointestinales/metabolismo , Neoplasias Gastrointestinales/patología , Humanos , Estructura Molecular , Relación Estructura-Actividad , Talidomida/síntesis química , Talidomida/química , Tiazoles/síntesis química , Tiazoles/química , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Increased PI 3-kinase (PI3K) signaling in pancreatic ductal adenocarcinoma (PDAC) correlates with poor prognosis, but the role of class I PI3K isoforms during its induction remains unclear. Using genetically engineered mice and pharmacological isoform-selective inhibitors, we found that the p110α PI3K isoform is a major signaling enzyme for PDAC development induced by a combination of genetic and nongenetic factors. Inactivation of this single isoform blocked the irreversible transition of exocrine acinar cells into pancreatic preneoplastic ductal lesions by oncogenic Kras and/or pancreatic injury. Hitting the other ubiquitous isoform, p110ß, did not prevent preneoplastic lesion initiation. p110α signaling through small GTPase Rho and actin cytoskeleton controls the reprogramming of acinar cells and regulates cell morphology in vivo and in vitro. Finally, p110α was necessary for pancreatic ductal cancers to arise from Kras-induced preneoplastic lesions by increasing epithelial cell proliferation in the context of mutated p53. Here we identify an in vivo context in which p110α cellular output differs depending on the epithelial transformation stage and demonstrate that the PI3K p110α is required for PDAC induced by oncogenic Kras, the key driver mutation of PDAC. These data are critical for a better understanding of the development of this lethal disease that is currently without efficient treatment.