RESUMO
Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.
Assuntos
Antineoplásicos/farmacologia , Terapia de Alvo Molecular , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/química , Animais , Antineoplásicos/química , Calorimetria , Linhagem Celular , Fibroblastos/metabolismo , Xenoenxertos , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras , Transdução de Sinais , Bibliotecas de Moléculas PequenasRESUMO
BACKGROUND & AIMS: The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but contradicts the current concept of one-size-fits-all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the nuclear factor of activated T cells (SMAD4-/-/NFATc1High). METHODS: Transcriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. RESULTS: Our findings determined the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. CONCLUSIONS: Our results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a mitogen-activated protein kinase signaling inhibition and gemcitabine combination therapy.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Gencitabina , Linhagem Celular Tumoral , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Smad3/metabolismoRESUMO
Proper Hedgehog (HH) signaling is essential for embryonic development, while aberrant HH signaling drives pediatric and adult cancers. HH signaling is frequently dysregulated in pancreatic cancer, yet its role remains controversial, with both tumor-promoting and tumor-restraining functions reported. Notably, the GLI family of HH transcription factors (GLI1, GLI2, GLI3), remain largely unexplored in pancreatic cancer. We therefore investigated the individual and combined contributions of GLI1-3 to pancreatic cancer progression. At pre-cancerous stages, fibroblast-specific Gli2/Gli3 deletion decreases immunosuppressive macrophage infiltration and promotes T cell infiltration. Strikingly, combined loss of Gli1/Gli2/Gli3 promotes macrophage infiltration, indicating that subtle changes in Gli expression differentially regulate immune infiltration. In invasive tumors, Gli2/Gli3 KO fibroblasts exclude immunosuppressive myeloid cells and suppress tumor growth by recruiting natural killer cells. Finally, we demonstrate that fibroblasts directly regulate macrophage and T cell migration through the expression of Gli-dependent cytokines. Thus, the coordinated activity of GLI1-3 directs the fibroinflammatory response throughout pancreatic cancer progression.
Assuntos
Proteínas Hedgehog , Neoplasias Pancreáticas , Adulto , Criança , Feminino , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Pancreáticas/genética , Gravidez , Proteína GLI1 em Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli3 com Dedos de Zinco/genéticaRESUMO
Alternative polyadenylation (APA) is a gene regulatory process that dictates mRNA 3'-UTR length, resulting in changes in mRNA stability and localization. APA is frequently disrupted in cancer and promotes tumorigenesis through altered expression of oncogenes and tumor suppressors. Pan-cancer analyses have revealed common APA events across the tumor landscape; however, little is known about tumor type-specific alterations that may uncover novel events and vulnerabilities. Here, we integrate RNA-sequencing data from the Genotype-Tissue Expression (GTEx) project and The Cancer Genome Atlas (TCGA) to comprehensively analyze APA events in 148 pancreatic ductal adenocarcinomas (PDACs). We report widespread, recurrent, and functionally relevant 3'-UTR alterations associated with gene expression changes of known and newly identified PDAC growth-promoting genes and experimentally validate the effects of these APA events on protein expression. We find enrichment for APA events in genes associated with known PDAC pathways, loss of tumor-suppressive miRNA binding sites, and increased heterogeneity in 3'-UTR forms of metabolic genes. Survival analyses reveal a subset of 3'-UTR alterations that independently characterize a poor prognostic cohort among PDAC patients. Finally, we identify and validate the casein kinase CSNK1A1 (also known as CK1alpha or CK1a) as an APA-regulated therapeutic target in PDAC. Knockdown or pharmacological inhibition of CSNK1A1 attenuates PDAC cell proliferation and clonogenic growth. Our single-cancer analysis reveals APA as an underappreciated driver of protumorigenic gene expression in PDAC via the loss of miRNA regulation.
Assuntos
Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/genética , Poliadenilação , Regiões 3' não Traduzidas , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Sítios de Ligação , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Caseína Quinase Ialfa/fisiologia , Proliferação de Células , Humanos , MicroRNAs/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Prognóstico , RNA-SeqRESUMO
Aberrant expression of CA125/MUC16 is associated with pancreatic ductal adenocarcinoma (PDAC) progression and metastasis. However, knowledge of the contribution of MUC16 to pancreatic tumorigenesis is limited. Here, we show that MUC16 expression is associated with disease progression, basal-like and squamous tumor subtypes, increased tumor metastasis, and short-term survival of PDAC patients. MUC16 enhanced tumor malignancy through the activation of AKT and GSK3ß oncogenic signaling pathways. Activation of these oncogenic signaling pathways resulted in part from increased interactions between MUC16 and epidermal growth factor (EGF)-type receptors, which were enhanced for aberrant glycoforms of MUC16. Treatment of PDAC cells with monoclonal antibody (mAb) AR9.6 significantly reduced MUC16-induced oncogenic signaling. mAb AR9.6 binds to a unique conformational epitope on MUC16, which is influenced by O-glycosylation. Additionally, treatment of PDAC tumor-bearing mice with either mAb AR9.6 alone or in combination with gemcitabine significantly reduced tumor growth and metastasis. We conclude that the aberrant expression of MUC16 enhances PDAC progression to an aggressive phenotype by modulating oncogenic signaling through ErbB receptors. Anti-MUC16 mAb AR9.6 blocks oncogenic activities and tumor growth and could be a novel immunotherapeutic agent against MUC16-mediated PDAC tumor malignancy.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antígeno Ca-125/genética , Carcinogênese/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Receptores ErbB/genética , Proteínas de Membrana/genética , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Animais , Anticorpos Monoclonais/farmacologia , Antígeno Ca-125/imunologia , Carcinogênese/imunologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Camundongos , Metástase Neoplásica , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Transdução de SinaisRESUMO
OBJECTIVE: Long-standing chronic pancreatitis is an established risk factor for pancreatic ductal adenocarcinoma (PDAC). Interleukin-1ß (IL-1ß) has been associated in PDAC with shorter survival. We employed murine models to investigate the mechanisms by which IL-1ß and chronic pancreatitis might contribute to PDAC progression. DESIGN: We crossed LSL-Kras+/G12D;Pdx1-Cre (KC) mice with transgenic mice overexpressing IL-1ß to generate KC-IL1ß mice, and followed them longitudinally. We used pancreatic 3D in vitro culture to assess acinar-to-ductal metaplasia formation. Immune cells were analysed by flow cytometry and immunohistochemical staining. B lymphocytes were adoptively transferred or depleted in Kras-mutant mice. B-cell infiltration was analysed in human PDAC samples. RESULTS: KC-IL1ß mice developed PDAC with liver metastases. IL-1ß treatment increased Kras+/G12D pancreatic spheroid formation. CXCL13 expression and B lymphocyte infiltration were increased in KC-IL1ß pancreata. Adoptive transfer of B lymphocytes from KC-IL1ß mice promoted tumour formation, while depletion of B cells prevented tumour progression in KC-IL1ß mice. B cells isolated from KC-IL1ß mice had much higher expression of PD-L1, more regulatory B cells, impaired CD8+ T cell activity and promoted tumorigenesis. IL-35 was increased in the KC-IL1ß pancreata, and depletion of IL-35 decreased the number of PD-L1+ B cells. Finally, in human PDAC samples, patients with PDAC with higher B-cell infiltration within tumours showed significantly shorter survival. CONCLUSION: We show here that IL-1ß promotes tumorigenesis in part by inducing an expansion of immune-suppressive B cells. These findings point to the growing significance of B suppressor cells in pancreatic tumorigenesis.
Assuntos
Linfócitos B/imunologia , Carcinoma Ductal Pancreático/etiologia , Tolerância Imunológica/imunologia , Neoplasias Pancreáticas/etiologia , Pancreatite/complicações , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/imunologia , Citometria de Fluxo , Interleucina-1beta/efeitos adversos , Camundongos , Camundongos Transgênicos , Neoplasias Pancreáticas/imunologia , Pancreatite/etiologia , Pancreatite/imunologiaRESUMO
BACKGROUND & AIMS: Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis. METHODS: We performed studies with LSL-KrasG12D/+;Ptf1aCre/+ mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA. RESULTS: Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D2 than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D2, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA. CONCLUSIONS: In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D2. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.
Assuntos
Carcinoma Ductal Pancreático/prevenção & controle , Transformação Celular Neoplásica/metabolismo , Pâncreas/metabolismo , Neoplasias Pancreáticas/prevenção & controle , Prostaglandina D2/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ceruletídeo , Modelos Animais de Doenças , Metabolismo Energético , Fibrose , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Camundongos Transgênicos , Mutação , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/metabolismo , Pancreatite/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Human leukocyte antigen (HLA) class I molecules present antigenic peptides to cytotoxic T cells, causing lysis of malignant cells. Transplantation biology studies have implicated HLA class I molecules in cell migration, but there has been little evidence presented that they influence cancer cell migration, a contributing factor in metastasis. In this study, we examined the effect of HLA-B on pancreatic cancer cell migration. HLA-B siRNA transfection increased the migration of the S2-013 pancreatic cancer cells but, in contrast, reduced migration of the PANC-1 and MIA PaCa-2 pancreatic cancer cell lines. Integrin molecules have previously been implicated in the upregulation of pancreatic cancer cell migration, and knockdown of HLA-B in S2-013 cells heightened the expression of integrin beta 1 (ITGB1), but in the PANC-1 and MIA PaCa-2 cells HLA-B knockdown diminished ITGB1 expression. A transmembrane sequence in an S2-013 HLA-B heavy chain matches a corresponding sequence in HLA-B in the BxPC-3 pancreatic cancer cell line, and knockdown of BxPC-3 HLA-B mimics the effect of S2-013 HLA-B knockdown on migration. In total, our findings indicate that HLA-B influences the expression of ITGB1 in pancreatic cancer cells, with concurrent distinctions in transmembrane sequences and effects on the migration of the cells.
Assuntos
Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Antígenos HLA-B/genética , Integrina beta1/genética , Pâncreas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Antígenos HLA-B/metabolismo , Humanos , Integrina alfa2/genética , Integrina alfa2/metabolismo , Integrina beta1/metabolismo , Especificidade de Órgãos , Pâncreas/patologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) is a highly metastatic disease with limited therapeutic options. Genome and transcriptome analyses have identified signalling pathways and cancer driver genes with implications in patient stratification and targeted therapy. However, these analyses were performed in bulk samples and focused on coding genes, which represent a small fraction of the genome. DESIGN: We developed a computational framework to reconstruct the non-coding transcriptome from cross-sectional RNA-Seq, integrating somatic copy number alterations (SCNA), common germline variants associated to PDA risk and clinical outcome. We validated the results in an independent cohort of paired epithelial and stromal RNA-Seq derived from laser capture microdissected human pancreatic tumours, allowing us to annotate the compartment specificity of their expression. We employed systems and experimental biology approaches to interrogate the function of epithelial long non-coding RNAs (lncRNAs) associated with genetic traits and clinical outcome in PDA. RESULTS: We generated a catalogue of PDA-associated lncRNAs. We showed that lncRNAs define molecular subtypes with biological and clinical significance. We identified lncRNAs in genomic regions with SCNA and single nucleotide polymorphisms associated with lifetime risk of PDA and associated with clinical outcome using genomic and clinical data in PDA. Systems biology and experimental functional analysis of two epithelial lncRNAs (LINC00673 and FAM83H-AS1) suggest they regulate the transcriptional profile of pancreatic tumour samples and PDA cell lines. CONCLUSIONS: Our findings indicate that lncRNAs are associated with genetic marks of pancreatic cancer risk, contribute to the transcriptional regulation of neoplastic cells and provide an important resource to design functional studies of lncRNAs in PDA.
Assuntos
Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , RNA Longo não Codificante/genética , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Marcadores Genéticos/genética , Mutação em Linhagem Germinativa , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Estimativa de Kaplan-Meier , Polimorfismo de Nucleotídeo Único , Prognóstico , RNA Neoplásico/genética , TranscriptomaRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) has among the highest stromal fractions of any cancer and this has complicated attempts at expression-based molecular classification. The goal of this work is to profile purified samples of human PDA epithelium and stroma and examine their respective contributions to gene expression in bulk PDA samples. DESIGN: We used laser capture microdissection (LCM) and RNA sequencing to profile the expression of 60 matched pairs of human PDA malignant epithelium and stroma samples. We then used these data to train a computational model that allowed us to infer tissue composition and generate virtual compartment-specific expression profiles from bulk gene expression cohorts. RESULTS: Our analysis found significant variation in the tissue composition of pancreatic tumours from different public cohorts. Computational removal of stromal gene expression resulted in the reclassification of some tumours, reconciling functional differences between different cohorts. Furthermore, we established a novel classification signature from a total of 110 purified human PDA stroma samples, finding two groups that differ in the extracellular matrix-associated and immune-associated processes. Lastly, a systematic evaluation of cross-compartment subtypes spanning four patient cohorts indicated partial dependence between epithelial and stromal molecular subtypes. CONCLUSION: Our findings add clarity to the nature and number of molecular subtypes in PDA, expand our understanding of global transcriptional programmes in the stroma and harmonise the results of molecular subtyping efforts across independent cohorts.
Assuntos
Adenocarcinoma/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Adenocarcinoma/genética , Adenocarcinoma/cirurgia , Carcinoma Ductal Pancreático/cirurgia , Estudos de Casos e Controles , Simulação por Computador , Matriz Extracelular/patologia , Perfilação da Expressão Gênica , Humanos , Microdissecção , Neoplasias Pancreáticas/cirurgia , Sensibilidade e EspecificidadeRESUMO
Epigenetic dysregulation is involved in the initiation and progression of many epithelial cancers. BMI1, a component of the polycomb protein family, plays a key role in these processes by controlling the histone ubiquitination and long-term repression of multiple genomic loci. BMI1 has previously been implicated in pancreatic homeostasis and the function of pancreatic cancer stem cells. However, no work has yet addressed its role in the early stages of pancreatic cancer development. Here, we show that BMI1 is required for the initiation of murine pancreatic neoplasia using a novel conditional knockout of Bmi1 in combination with a Kras(G12D)-driven pancreatic cancer mouse model. We also demonstrate that the requirement for Bmi1 in pancreatic carcinogenesis is independent of the Ink4a/Arf locus and at least partially mediated by dysregulation of reactive oxygen species. Our data provide new evidence of the importance of this epigenetic regulator in the genesis of pancreatic cancer.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND & AIMS: Metaplasias often have characteristics of developmentally related tissues. Pancreatic metaplastic ducts are usually associated with pancreatitis and pancreatic ductal adenocarcinoma. The tuft cell is a chemosensory cell that responds to signals in the extracellular environment via effector molecules. Commonly found in the biliary tract, tuft cells are absent from normal murine pancreas. Using the aberrant appearance of tuft cells as an indicator, we tested if pancreatic metaplasia represents transdifferentiation to a biliary phenotype and what effect this has on pancreatic tumorigenesis. METHODS: We analyzed pancreatic tissue and tumors that developed in mice that express an activated form of Kras (Kras(LSL-G12D/+);Ptf1a(Cre/+) mice). Normal bile duct, pancreatic duct, and tumor-associated metaplasias from the mice were analyzed for tuft cell and biliary progenitor markers, including SOX17, a transcription factor that regulates biliary development. We also analyzed pancreatic tissues from mice expressing transgenic SOX17 alone (ROSA(tTa/+);Ptf1(CreERTM/+);tetO-SOX17) or along with activated Kras (ROSAtT(a/+);Ptf1a(CreERTM/+);tetO-SOX17;Kras(LSL-G12D;+)). RESULTS: Tuft cells were frequently found in areas of pancreatic metaplasia, decreased throughout tumor progression, and absent from invasive tumors. Analysis of the pancreatobiliary ductal systems of mice revealed tuft cells in the biliary tract but not the normal pancreatic duct. Analysis for biliary markers revealed expression of SOX17 in pancreatic metaplasia and tumors. Pancreas-specific overexpression of SOX17 led to ductal metaplasia along with inflammation and collagen deposition. Mice that overexpressed SOX17 along with Kras(G12D) had a greater degree of transformed tissue compared with mice expressing only Kras(G12D). Immunofluorescence analysis of human pancreatic tissue arrays revealed the presence of tuft cells in metaplasia and early-stage tumors, along with SOX17 expression, consistent with a biliary phenotype. CONCLUSIONS: Expression of Kras(G12D) and SOX17 in mice induces development of metaplasias with a biliary phenotype containing tuft cells. Tuft cells express a number of tumorigenic factors that can alter the microenvironment. Expression of SOX17 induces pancreatitis and promotes Kras(G12D)-induced tumorigenesis in mice.
Assuntos
Ductos Biliares/citologia , Carcinoma Ductal Pancreático/patologia , Transformação Celular Neoplásica/patologia , Proteínas HMGB/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/patologia , Lesões Pré-Cancerosas/patologia , Fatores de Transcrição SOXF/metabolismo , Animais , Ductos Biliares/metabolismo , Carcinoma Ductal Pancreático/complicações , Carcinoma Ductal Pancreático/metabolismo , Transformação Celular Neoplásica/metabolismo , Humanos , Metaplasia/complicações , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos , Camundongos Transgênicos , Ductos Pancreáticos/citologia , Ductos Pancreáticos/metabolismo , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/metabolismo , Pancreatite/metabolismo , Lesões Pré-Cancerosas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de SinaisRESUMO
The broad application of noninvasive imaging has transformed preclinical cancer research, providing a powerful means to measure dynamic processes in living animals. While imaging technologies are routinely used to monitor tumor growth in model systems, their greatest potential lies in their ability to answer fundamental biological questions. Here we present the broad range of potential imaging applications according to the needs of a cancer biologist with a focus on some of the common biological processes that can be used to visualize and measure. Topics include imaging metastasis; biophysical properties such as perfusion, diffusion, oxygenation, and stiffness; imaging the immune system and tumor microenvironment; and imaging tumor metabolism. We also discuss the general ability of each approach and the level of training needed to both acquire and analyze images. The overall goal is to provide a practical guide for cancer biologists interested in answering biological questions with preclinical imaging technologies.
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Pancreatic ductal adenocarcinoma-(PDAC) needs innovative approaches due to its 12% 5-year survival despite current therapies. We show marked sensitivity of pancreatic cancer cells to the combination of a novel eIF4A inhibitor, des-methyl pateamine A (DMPatA), and a histone deacetylase inhibitor, romidepsin, inducing epigenetic reprogramming as an innovative therapeutic strategy. Exploring the mechanistic activity of this combination showed that with a short duration of romidepsin at low doses, robust acetylation persisted up to 48h with the combination, while histone acetylation rapidly faded with monotherapy. This represents an unexpected mechanism of action against PDAC cells that triggers transcriptional overload, metabolic stress, and augmented DNA damage. Structurally different class I HDAC inhibitors exhibit the same hyperacetylation patterns when co-administered with DMPatA, suggesting a class effect. We show efficacy of this combination regimen against tumor growth in a MIA PaCa-2 xenograft model of PDAC with persistent hyperacetylation confirmed in tumor samples. STATEMENT OF SIGNIFICANCE: Pancreatic ductal adenocarcinoma, a significant clinical challenge, could benefit from the latent potential of epigenetic therapies like HDAC inhibitors-(HDIs), typically limited to hematological malignancies. Our study shows that a synergistic low dose combination of HDIs with an eIF4A-inhibitor in pancreatic cancer models results in marked pre-clinical efficacy, offering a promising new treatment strategy.
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Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges for effective treatment, with systemic chemotherapy often proving inadequate due to poor drug delivery and the tumor's immunosuppressive microenvironment. Engineered bacteria present a novel approach to target PDAC, leveraging their ability to colonize tumors and deliver therapeutic payloads. Here, we engineered probiotic Escherichia coli Nissle 1917 (EcN) to produce the pore-forming Theta toxin (Nis-Theta) and evaluated its efficacy in a preclinical model of PDAC. Probiotic administration resulted in selective colonization of tumor tissue, leading to improved overall survival compared to standard chemotherapy. Moreover, this strain exhibited cytotoxic effects on both primary and distant tumor lesions while sparing normal tissues. Importantly, treatment also modulated the tumor microenvironment by increasing anti-tumor immune cell populations and reducing immunosuppressive markers. These findings demonstrate the potential of engineered probiotic bacteria as a safe and effective therapeutic approach for PDAC, offering promise for improved patient outcomes.
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The sparse vascularity of pancreatic ductal adenocarcinoma (PDAC) presents a mystery: What prevents this aggressive malignancy from undergoing neoangiogenesis to counteract hypoxia and better support growth? An incidental finding from prior work on paracrine communication between malignant PDAC cells and fibroblasts revealed that inhibition of the Hedgehog (HH) pathway partially relieved angiosuppression, increasing tumor vascularity through unknown mechanisms. Initial efforts to study this phenotype were hindered by difficulties replicating the complex interactions of multiple cell types in vitro. Here we identify a cascade of paracrine signals between multiple cell types that act sequentially to suppress angiogenesis in PDAC. Malignant epithelial cells promote HH signaling in fibroblasts, leading to inhibition of noncanonical WNT signaling in fibroblasts and epithelial cells, thereby limiting VEGFR2-dependent activation of endothelial hypersprouting. This cascade was elucidated using human and murine PDAC explant models, which effectively retain the complex cellular interactions of native tumor tissues. SIGNIFICANCE: We present a key mechanism of tumor angiosuppression, a process that sculpts the physiologic, cellular, and metabolic environment of PDAC. We further present a computational and experimental framework for the dissection of complex signaling cascades that propagate among multiple cell types in the tissue environment. This article is featured in Selected Articles from This Issue, p. 201.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Proteínas Hedgehog/genética , Neoplasias Pancreáticas/patologia , Fator A de Crescimento do Endotélio VascularRESUMO
Traditional 2D/3D co-culture models typically do not reflect the cellular heterogeneity of pancreatic ductal adenocarcinoma (PDAC) tumors, while in vivo models can be slow and ill-suited to mechanistic investigations. Here, we present a protocol for culturing murine PDAC explants and a corresponding human PDAC model using tissue slice explants. We describe steps for sponge production, preparation of media and materials, tissue collection, and sectioning. We then detail procedures for explant plating, daily culture, and collection of samples.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Camundongos , Animais , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Ductos Pancreáticos , Técnicas de CoculturaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease characterized by its metastatic potential and chemoresistance. These traits are partially attributable to the highly tumorigenic pancreatic cancer stem cells (PaCSCs). Interestingly, these cells show unique features in order to sustain their identity and functionality, some of them amenable for therapeutic intervention. Screening of phospho-receptor tyrosine kinases revealed that PaCSCs harbored increased activation of anaplastic lymphoma kinase (ALK). We subsequently demonstrated that oncogenic ALK signaling contributes to tumorigenicity in PDAC patient-derived xenografts (PDXs) by promoting stemness through ligand-dependent activation. Indeed, the ALK ligands midkine (MDK) or pleiotrophin (PTN) increased self-renewal, clonogenicity and CSC frequency in several in vitro local and metastatic PDX models. Conversely, treatment with the clinically-approved ALK inhibitors Crizotinib and Ensartinib decreased PaCSC content and functionality in vitro and in vivo, by inducing cell death. Strikingly, ALK inhibitors sensitized chemoresistant PaCSCs to Gemcitabine, as the most used chemotherapeutic agent for PDAC treatment. Consequently, ALK inhibition delayed tumor relapse after chemotherapy in vivo by effectively decreasing the content of PaCSCs. In summary, our results demonstrate that targeting the MDK/PTN-ALK axis with clinically-approved inhibitors impairs in vivo tumorigenicity and chemoresistance in PDAC suggesting a new treatment approach to improve the long-term survival of PDAC patients.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Quinase do Linfoma Anaplásico , Resistencia a Medicamentos Antineoplásicos , Recidiva Local de Neoplasia , Receptores Proteína Tirosina Quinases , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Linhagem Celular Tumoral , Neoplasias PancreáticasRESUMO
BACKGROUND: Efficient presentation of mutant peptide fragments by the human leukocyte antigen class I (HLA-I) genes is necessary for immune-mediated killing of cancer cells. According to recent reports, patient HLA-I genotypes can impact the efficacy of cancer immunotherapy, and the somatic loss of HLA-I heterozygosity has been established as a factor in immune evasion. While global deregulated expression of HLA-I has also been reported in different tumor types, the role of HLA-I allele-specific expression loss - that is, the preferential RNA expression loss of specific HLA-I alleles - has not been fully characterized in cancer. METHODS: Here, we use RNA and whole-exome sequencing data to quantify HLA-I allele-specific expression (ASE) in cancer using our novel method arcasHLA-quant. RESULTS: We show that HLA-I ASE loss in at least one of the three HLA-I genes is a pervasive phenomenon across TCGA tumor types. In pancreatic adenocarcinoma, tumor-specific HLA-I ASE loss is associated with decreased overall survival specifically in the basal-like subtype, a finding that we validated in an independent cohort through laser-capture microdissection. Additionally, we show that HLA-I ASE loss is associated with poor immunotherapy outcomes in metastatic melanoma through retrospective analyses. CONCLUSIONS: Together, our results highlight the prevalence of HLA-I ASE loss and provide initial evidence of its clinical significance in cancer prognosis and immunotherapy treatment.