RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal malignancy with extremely poor patient survival rates. A key reason for the poor prognosis is the lack of effective diagnostic tools to detect the disease at curable, premetastatic stages. Tumor surgical resection is PDAC's first-line treatment, however distinguishing between cancerous and healthy tissue with current imaging tools remains a challenge. In this work, we report a DOTA-based fluorescent probe targeting plectin-1 for imaging PDAC with high specificity. To enable heterogeneous functionalization of the DOTA-core with multiple targeting peptide units and the fluorophore, a novel, fully clickable synthetic route that proceeds in one pot was developed. Extensive validation of the probe set the stage for PDAC detection in mice and human tissue. Altogether, these findings may pave the way for improved clinical understanding and early detection of PDAC progression as well as more accurate resection criteria.
Assuntos
Meios de Contraste , Compostos Heterocíclicos com 1 Anel , Neoplasias Pancreáticas , Plectina , Humanos , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/patologia , Plectina/metabolismo , Animais , Meios de Contraste/química , Camundongos , Compostos Heterocíclicos com 1 Anel/química , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/patologia , Imagem ÓpticaRESUMO
Solid cancers like pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer, frequently exploit nerves for rapid dissemination. This neural invasion (NI) is an independent prognostic factor in PDAC, but insufficiently modeled in genetically engineered mouse models (GEMM) of PDAC. Here, we systematically screened for human-like NI in Europe's largest repository of GEMM of PDAC, comprising 295 different genotypes. This phenotype screen uncovered 2 GEMMs of PDAC with human-like NI, which are both characterized by pancreas-specific overexpression of transforming growth factor α (TGF-α) and conditional depletion of p53. Mechanistically, cancer-cell-derived TGF-α upregulated CCL2 secretion from sensory neurons, which induced hyperphosphorylation of the cytoskeletal protein paxillin via CCR4 on cancer cells. This activated the cancer migration machinery and filopodia formation toward neurons. Disrupting CCR4 or paxillin activity limited NI and dampened tumor size and tumor innervation. In human PDAC, phospho-paxillin and TGF-α-expression constituted strong prognostic factors. Therefore, we believe that the TGF-α-CCL2-CCR4-p-paxillin axis is a clinically actionable target for constraining NI and tumor progression in PDAC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo , Paxilina/genética , Paxilina/metabolismo , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/metabolismo , Fenótipo , Linhagem Celular Tumoral , Neoplasias PancreáticasRESUMO
Over 90% of pancreatic cancers present mutations in KRAS, one of the most common oncogenic drivers overall. Currently, most KRAS mutant isoforms cannot be targeted directly. Moreover, targeting single RAS downstream effectors induces adaptive resistance mechanisms. We report here on the combined inhibition of SHP2, upstream of KRAS, using the allosteric inhibitor RMC-4550 and of ERK, downstream of KRAS, using LY3214996. This combination shows synergistic anti-cancer activity in vitro, superior disruption of the MAPK pathway, and increased apoptosis induction compared with single-agent treatments. In vivo, we demonstrate good tolerability and efficacy of the combination, with significant tumor regression in multiple pancreatic ductal adenocarcinoma (PDAC) mouse models. Finally, we show evidence that 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) can be used to assess early drug responses in animal models. Based on these results, we will investigate this drug combination in the SHP2 and ERK inhibition in pancreatic cancer (SHERPA; ClinicalTrials.gov: NCT04916236) clinical trial, enrolling patients with KRAS-mutant PDAC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Camundongos , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Ensaios Clínicos como Assunto , Neoplasias PancreáticasRESUMO
miRNAs have crucial functions in many biological processes and are candidate biomarkers of disease. Here, we show that miR-216a is a conserved, pancreas-specific miRNA with important roles in pancreatic islet and acinar cells. Deletion of miR-216a in mice leads to a reduction in islet size, ß-cell mass, and insulin levels. Single-cell RNA sequencing reveals a subpopulation of ß-cells with upregulated acinar cell markers under a high-fat diet. miR-216a is induced by TGF-ß signaling, and inhibition of miR-216a increases apoptosis and decreases cell proliferation in pancreatic cells. Deletion of miR-216a in the pancreatic cancer-prone mouse line KrasG12D;Ptf1aCreER reduces the propensity of pancreatic cancer precursor lesions. Notably, circulating miR-216a levels are elevated in both mice and humans with pancreatic cancer. Collectively, our study gives insights into how ß-cell mass and acinar cell growth are modulated by a pancreas-specific miRNA and also suggests miR-216a as a potential biomarker for diagnosis of pancreatic diseases.
Assuntos
Progressão da Doença , Deleção de Genes , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Animais , Apoptose , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular , Dieta Hiperlipídica , Humanos , Secreção de Insulina , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/metabolismo , Especificidade de Órgãos , RatosAssuntos
Integrases/genética , Herança Paterna , Recombinação Genética , Fatores de Transcrição/genética , Animais , Feminino , Genótipo , Humanos , Integrases/metabolismo , Masculino , Camundongos Transgênicos , Mutação , Fenótipo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-rel/genética , Proteínas Proto-Oncogênicas c-rel/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismoRESUMO
BACKGROUND & AIMS: The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. METHODS: Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing in vivo and in vitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. RESULTS: Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. CONCLUSIONS: We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches.
Assuntos
Proteína 3 do Linfoma de Células B/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Proteína 3 do Linfoma de Células B/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundário , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Metabolismo Energético , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Carga Tumoral , Células Tumorais CultivadasRESUMO
Several studies have suggested an oncogenic role for the neural Wiskott-Aldrich syndrome protein (N-WASP, encoded by the Wasl gene), but thus far, little is known about its function in pancreatic ductal adenocarcinoma (PDAC). In this study, we performed in silico analysis of WASL expression in PDAC patients and found a correlation between low WASL expression and prolonged survival. To clarify the role of Wasl in pancreatic carcinogenesis, we used 2 oncogenic Kras-based PDAC mouse models with pancreas-specific Wasl deletion. In line with human data, both mouse models had an increased survival benefit due to either impaired tumor development in the presence of the tumor suppressor Trp53 or the delayed tumor progression and senescent phenotype upon genetic ablation of Trp53. Mechanistically, loss of Wasl resulted in cell-autonomous senescence through displacement of the N-WASP binding partners WASP-interacting protein (WIP) and p120ctn; vesicular accumulation of GSK3ß, as well as YAP1 and phosphorylated ß-catenin, which are components of the destruction complex; and upregulation of Cdkn1a(p21), a master regulator of senescence. Our findings, thus, indicate that Wasl functions in an oncogenic manner in PDAC by promoting the deregulation of the p120-catenin/ß-catenin/p21 pathway. Therefore, strategies to reduce N-WASP activity might improve the survival outcomes of PDAC patients.
Assuntos
Neoplasias Experimentais/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/deficiência , Animais , Humanos , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Pancreatic cancer is one of the deadliest cancer types urgently requiring effective therapeutic strategies. Autophagy occurs in several compartments of pancreatic cancer tissue including cancer cells, cancer associated fibroblasts, and immune cells where it can be subjected to a multitude of stimulatory and inhibitory signals fine-tuning its activity. Therefore, the effects of autophagy on pancreatic carcinogenesis and progression differ in a stage and context dependent manner. In the initiation stage autophagy hinders development of preneoplastic lesions; in the progression stage however, autophagy promotes tumor growth. This double-edged action of autophagy makes it a hard therapeutic target. Indeed, autophagy inhibitors have not yet shown survival improvements in clinical trials, indicating a need for better evaluation of existing results and smarter targeting techniques. Clearly, the role of autophagy in pancreatic cancer is complex and many aspects have to be considered when moving from the bench to the bedside.
Assuntos
Autofagia , Neoplasias Pancreáticas/patologia , Pesquisa Translacional Biomédica , Animais , Ensaios Clínicos como Assunto , Humanos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/terapia , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
Pancreatic ductal adenocarcinoma (PDAC) will soon belong to the top three cancer killers. The only approved specific PDAC therapy targets the epidermal growth factor receptor (EGFR). Although EGFR is a crucial player in PDAC development, EGFR-based therapy is disappointing. In this study, we evaluated the role of the EGFR ligand betacellulin (BTC) in PDAC. The expression of BTC was investigated in human pancreatic cancer specimen. Then, we generated a BTC knockout mouse model by CRISPR/Cas9 technology and a BTC overexpression model. Both models were crossed with the Ptf1aCre/+ ;KRASG12D/+ (KC) mouse model (B-/- KC or BKC, respectively). In addition, EGFR, ERBB2, and ERBB4 were investigated by the pancreas-specific deletion of each receptor using the Cre-loxP system. Tumor initiation and progression were analyzed in all mouse lines, and the underlying molecular biology of PDAC was investigated at different time points. BTC is expressed in human and murine PDAC. B-/- KC mice showed a decelerated PDAC progression, associated with decreased EGFR activation. BKC mice developed severe PDAC with a poor survival rate. The dramatically increased BTC-mediated tumor burden was EGFR-dependent, but also ERBB4 and ERBB2 were involved in PDAC development or progression, as depletion of EGFR, ERBB2, or ERBB4 significantly improved the survival rate of BTC-mediated PDAC. BTC increases PDAC tumor burden dramatically by enhanced RAS activation. EGFR signaling, ERBB2 signaling, and ERBB4 signaling are involved in accelerated PDAC development mediated by BTC indicating that targeting the whole ERBB family, instead of a single receptor, is a promising strategy for the development of future PDAC therapies.
Assuntos
Adenocarcinoma/metabolismo , Betacelulina/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-4/metabolismo , Transdução de Sinais , Animais , Peso Corporal , Receptores ErbB/metabolismo , Humanos , Camundongos Transgênicos , Pâncreas/metabolismo , Pâncreas/patologia , Fenótipo , Fosforilação , Carga Tumoral , Proteínas ras/metabolismo , Neoplasias PancreáticasRESUMO
BACKGROUND: The EGFR ligand betacellulin (BTC) has been previously shown to protect mice against experimentally induced acute pancreatitis (AP). BTC binds both autonomous ERBB receptors EGFR and ERBB4. In this study, we evaluated the mechanism underlying the protection from AP-associated inflammation in detail. METHODS: AP was induced with cerulein or L-arginine and investigated in a pancreas-specific ERBB4 knockout and in an EGFR knockdown mouse model (EgfrWa5/+). Pancreatitis was evaluated by scoring inflammation, necrosis, and edema, while microarrays were performed to analyze alterations in the transcriptome between mice with AP and animals which were protected against AP. The intracellular domain (ICD) of ERBB4 was analyzed in different cell compartments. RESULTS: While the pancreas of BTC transgenic mice in the background of EgfrWa5/+ is still protected against AP, the BTC-mediated protection is no longer present in the absence of ERBB4. We further demonstrate that BTC activates the ICD of ERBB4, and increases the expression of the extracellular matrix (ECM) proteins periostin and matrix gla protein as well as the ECM modulators matrix metalloproteinases 2 and 3, but only in the presence of ERBB4. Notably, the increased expression of these proteins is not accompanied by an increased ECM amount. CONCLUSIONS: These findings suggest that BTC derivates, as a drug, or the ERBB4 receptor, as a druggable target protein, could play an important role in modulating the course of AP and even prevent AP in humans.
Assuntos
Betacelulina/farmacologia , Receptores ErbB/genética , Pancreatite/prevenção & controle , Receptor ErbB-4/genética , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pancreatite/genéticaRESUMO
BACKGROUND: Pain due to pancreatic cancer/PCa or chronic pancreatitis/CP, is notoriously resistant to the strongest pain medications. Here, we aimed at deciphering the specific molecular mediators of pain at surgical-stage pancreatic disease and to discover novel translational targets. METHODS: We performed a systematic, quantitative analysis of the neurotransmitter/neuroenzmye profile within intrapancreatic nerves of CP and PCa patients. Ex vivo neuronal cultures treated with human pancreatic extracts, conditional genetically engineered knockout mouse models of PCa and CP, and the cerulein-induced CP model were employed to explore the therapeutic potential of the identified targets. FINDINGS: We identified a unique enrichment of neuronal nitric-oxide-synthase (nNOS) in the pancreatic nerves of CP patients with increasing pain severity. Employment of ex vivo neuronal cultures treated with pancreatic tissue extracts of CP patients, and brain-derived-neurotrophic-factor-deficient (BDNF+/-) mice revealed neuronal enrichment of nNOS to be a consequence of BDNF loss in the progressively destroyed pancreatic tissue. Mechanistically, nNOS upregulation in sensory neurons was induced by tryptase secreted from perineural mast cells. In a head-to-head comparison of several genetically induced, painless mouse models of PCa (KPC, KC mice) or CP (Ptf1a-Cre;Atg5fl/fl) against the hypersecretion/cerulein-induced, painful CP mouse model, we show that a similar nNOS enrichment is present in the painful cerulein-CP model, but absent in painless genetic models. Consequently, mice afflicted with painful cerulein-induced CP could be significantly relieved upon treatment with the specific nNOS inhibitor NPLA. INTERPRETATION: We propose nNOS inhibition as a novel strategy to treat the unbearable pain in CP. FUND: Deutsche Forschungsgemeinschaft/DFG (DE2428/3-1 and 3-2).
Assuntos
Neuralgia/diagnóstico , Neuralgia/etiologia , Óxido Nítrico Sintase Tipo I/metabolismo , Pancreatite Crônica/complicações , Pancreatite Crônica/metabolismo , Adulto , Animais , Biomarcadores , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neuralgia/tratamento farmacológico , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/cirurgia , Pancreatite Crônica/cirurgiaRESUMO
BACKGROUND AND AIMS: Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy related 5 (Atg5) protein on pancreatic tumor formation and progression. METHODS: We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/-;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times. RESULTS: A5+/-;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/-;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/-;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/-;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time. CONCLUSIONS: In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.
Assuntos
Proteína 5 Relacionada à Autofagia/metabolismo , Autofagia , Carcinoma Ductal Pancreático/metabolismo , Movimento Celular , Neoplasias Pancreáticas/metabolismo , Animais , Proteína 5 Relacionada à Autofagia/deficiência , Proteína 5 Relacionada à Autofagia/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/prevenção & controle , Carcinoma Ductal Pancreático/secundário , Catepsinas/genética , Catepsinas/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Genes ras , Heterozigoto , Homozigoto , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/prevenção & controle , Transdução de Sinais , Carga Tumoral , Células Tumorais CultivadasRESUMO
The ubiquitously expressed non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, is involved in signal transduction downstream of multiple growth factor, cytokine and integrin receptors1. Its requirement for complete RAS-MAPK activation and its role as a negative regulator of JAK-STAT signaling have established SHP2 as an essential player in oncogenic signaling pathways1-7. Recently, a novel potent allosteric SHP2 inhibitor was presented as a viable therapeutic option for receptor tyrosine kinase-driven cancers, but was shown to be ineffective in KRAS-mutant tumor cell lines in vitro8. Here, we report a central and indispensable role for SHP2 in oncogenic KRAS-driven tumors. Genetic deletion of Ptpn11 profoundly inhibited tumor development in mutant KRAS-driven murine models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. We provide evidence for a critical dependence of mutant KRAS on SHP2 during carcinogenesis. Deletion or inhibition of SHP2 in established tumors delayed tumor progression but was not sufficient to achieve tumor regression. However, SHP2 was necessary for resistance mechanisms upon blockade of MEK. Synergy was observed when both SHP2 and MEK were targeted, resulting in sustained tumor growth control in murine and human patient-derived organoids and xenograft models of pancreatic ductal adenocarcinoma and non-small-cell lung cancer. Our data indicate the clinical utility of dual SHP2/MEK inhibition as a targeted therapy approach for KRAS-mutant cancers.
Assuntos
Mutação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Tirosina Fosfatase não Receptora Tipo 11/deficiênciaRESUMO
Pancreatic ductal adenocarcinomas (PDAC) harbor recurrent functional mutations of the master DNA damage response kinase ATM, which has been shown to accelerate tumorigenesis and epithelial-mesenchymal transition. To study how ATM deficiency affects genome integrity in this setting, we evaluated the molecular and functional effects of conditional Atm deletion in a mouse model of PDAC. ATM deficiency was associated with increased mitotic defects, recurrent genomic rearrangements, and deregulated DNA integrity checkpoints, reminiscent of human PDAC. We hypothesized that altered genome integrity might allow synthetic lethality-based options for targeted therapeutic intervention. Supporting this possibility, we found that the PARP inhibitor olaparib or ATR inhibitors reduced the viability of PDAC cells in vitro and in vivo associated with a genotype-selective increase in apoptosis. Overall, our results offered a preclinical mechanistic rationale for the use of PARP and ATR inhibitors to improve treatment of ATM-mutant PDAC. Cancer Res; 77(20); 5576-90. ©2017 AACR.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/enzimologia , Dano ao DNA , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/enzimologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Fluoruracila/farmacologia , Expressão Gênica , Instabilidade Genômica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos SCID , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , GencitabinaRESUMO
Stimulation with lipopolysaccharide (LPS; endotoxin) not only causes rapid production of proinflammatory cytokines, but also induces a state of LPS hypo-responsiveness to a second LPS stimulation (endotoxin tolerance (ET)). Murine bone marrow-derived MCs (BMMCs) and peritoneal MCs (PMCs) developed ET as shown by an abrogated production of Il6/Tnf RNAs and IL-6/TNF-α proteins. In naive BMMCs, LPS stimulation induced a transient decline in the trimethylation of lysine 9 of the core histone H3 (H3K9me3), a suppressive chromatin mark, at the Il6/Tnf promoters, which correlated with p50(NFκB) and p65(NFκB) binding. Both demethylation and NFκB binding were abrogated in tolerant cells. In addition, cytosolic NFκB activation was suppressed in tolerant BMMCs. Intriguingly, antigen stimulation of naive and tolerant MCs induced comparable production of Il6/Tnf and IL-6/TNF-α, although ET also affected antigen-triggered activation of NFκB; pharmacological analysis indicated the importance of Ca2+-dependent transcription in this respect. In macrophages, the IκB member BCL3 is induced by LPS and known to be involved in ET, which was not corroborated comparing wild-type and Bcl3-deficient BMMCs. Interestingly, Bcl3-deficient PMCs produce markedly increased amounts of IL-6/TNF-α after LPS stimulation. Collectively, ET in MCs is BCL3-independent, however, in PMCs, BCL3 negatively regulates immediate LPS-induced cytokine production and quantitatively affects ET.
Assuntos
Endotoxinas/imunologia , Imunoglobulina E/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Transdução de Sinais , Fatores de Transcrição/deficiência , Animais , Proteína 3 do Linfoma de Células B , Células da Medula Óssea/metabolismo , Citocinas/genética , Citocinas/metabolismo , Técnicas de Inativação de Genes , Histonas/metabolismo , Tolerância Imunológica , Mediadores da Inflamação , Metilação , Camundongos , Camundongos Knockout , Modelos Biológicos , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Receptor 4 Toll-Like/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) cells (PCC) have an exceptional propensity to metastasize early into intratumoral, chemokine-secreting nerves. However, we hypothesized the opposite process, that precancerous pancreatic cells secrete chemokines that chemoattract Schwann cells (SC) of nerves and thus induce ready-to-use routes of dissemination in early carcinogenesis. Here we show a peculiar role for the chemokine CXCL12 secreted in early PDAC and for its receptors CXCR4/CXCR7 on SC in the initiation of neural invasion in the cancer precursor stage and the resulting delay in the onset of PDAC-associated pain. SC exhibited cancer- or hypoxia-induced CXCR4/CXCR7 expression in vivo and in vitro and migrated toward CXCL12-expressing PCC. Glia-specific depletion of CXCR4/CXCR7 in mice abrogated the chemoattraction of SC to PCC. PDAC mice with pancreas-specific CXCL12 depletion exhibited diminished SC chemoattraction to pancreatic intraepithelial neoplasia and increased abdominal hypersensitivity caused by augmented spinal astroglial and microglial activity. In PDAC patients, reduced CXCR4/CXCR7 expression in nerves correlated with increased pain. Mechanistically, upon CXCL12 exposure, SC down-regulated the expression of several pain-associated targets. Therefore, PDAC-derived CXCL12 seems to induce tumor infiltration by SC during early carcinogenesis and to attenuate pain, possibly resulting in delayed diagnosis in PDAC.
Assuntos
Carcinoma Ductal Pancreático/patologia , Quimiocina CXCL12/metabolismo , Quimiotaxia/fisiologia , Dor/prevenção & controle , Neoplasias Pancreáticas/patologia , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Células de Schwann/fisiologia , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos TransgênicosRESUMO
Tumor suppression that is mediated by oncogene-induced senescence (OIS) is considered to function as a safeguard during development of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms that regulate OIS in PDAC are poorly understood. Here, we have determined that nuclear RelA reinforces OIS to inhibit carcinogenesis in the Kras mouse model of PDAC. Inactivation of RelA accelerated pancreatic lesion formation in Kras mice by abrogating the senescence-associated secretory phenotype (SASP) gene transcription signature. Using genetic and pharmacological tools, we determined that RelA activation promotes OIS via elevation of the SASP factor CXCL1 (also known as KC), which activates CXCR2, during pancreatic carcinogenesis. In Kras mice, pancreas-specific inactivation of CXCR2 prevented OIS and was correlated with increased tumor proliferation and decreased survival. Moreover, reductions in CXCR2 levels were associated with advanced neoplastic lesions in tissue from human pancreatic specimens. Genetically disabling OIS in Kras mice caused RelA to promote tumor proliferation, suggesting a dual role for RelA signaling in pancreatic carcinogenesis. Taken together, our data suggest a pivotal role for RelA in regulating OIS in preneoplastic lesions and implicate the RelA/CXCL1/CXCR2 axis as an essential mechanism of tumor surveillance in PDAC.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Senescência Celular , Quimiocina CXCL1/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores de Interleucina-8B/metabolismo , Fator de Transcrição RelA/metabolismo , Proteínas ras/genética , Animais , Carcinogênese , Carcinoma Ductal Pancreático/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes ras , Masculino , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Oncogenes , Neoplasias Pancreáticas/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Proteínas ras/metabolismoRESUMO
BACKGROUND & AIMS: One treatment strategy for pancreatic ductal adenocarcinoma is to modify, rather than deplete, the tumor stroma. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) is associated with progression of pancreatic and other solid tumors. We investigated whether loss of P53 function contributes to persistent activation of STAT3 and modification of the pancreatic tumor stroma in patients and mice. METHODS: Stat3, Il6st (encodes gp130), or Trp53 were disrupted, or a mutant form of P53 (P53R172H) or transgenic sgp130 were expressed, in mice that developed pancreatic tumors resulting from expression of activated KRAS (KrasG12D, KC mice). Pancreata were collected and analyzed by immunohistochemistry, in situ hybridization, quantitative reverse-transcription polymerase chain reaction (qPCR), or immunoblot assays; fluorescence-activated cell sorting was performed to identify immune cells. We obtained frozen pancreatic tumor specimens from patients and measured levels of phosphorylated STAT3 and P53 by immunohistochemistry; protein levels were associated with survival using Kaplan-Meier analyses. We measured levels of STAT3, P53, ligands for gp130, interleukin 6, cytokines, sonic hedgehog signaling, STAT3 phosphorylation (activation), and accumulation of reactive oxygen species in primary pancreatic cells from mice. Mice with pancreatic tumors were given gemcitabine and a Janus kinase 2 (JAK2) inhibitor; tumor growth was monitored by 3-dimensional ultrasound. RESULTS: STAT3 was phosphorylated constitutively in pancreatic tumor cells from KC mice with loss or mutation of P53. Tumor cells of these mice accumulated reactive oxygen species and had lower activity of the phosphatase SHP2 and prolonged phosphorylation of JAK2 compared with tumors from KC mice with functional P53. These processes did not require the gp130 receptor. Genetic disruption of Stat3 in mice, or pharmacologic inhibitors of JAK2 or STAT3 activation, reduced fibrosis and the numbers of pancreatic stellate cells in the tumor stroma and altered the types of immune cells that infiltrated tumors. Mice given a combination of gemcitabine and a JAK2 inhibitor formed smaller tumors and survived longer than mice given control agents; the tumor stroma had fewer activated pancreatic stellate cells, lower levels of periostin, and alterations in collagen production and organization. Phosphorylation of STAT3 correlated with P53 mutation and features of infiltrating immune cells in human pancreatic tumors. Patients whose tumors had lower levels of phosphorylated STAT3 and functional P53 had significantly longer survival times than patients with high levels of phosphorylated STAT3 and P53 mutation. CONCLUSIONS: In pancreatic tumors of mice, loss of P53 function activates JAK2-STAT3 signaling, which promotes modification of the tumor stroma and tumor growth and resistance to gemcitabine. In human pancreatic tumors, STAT3 phosphorylation correlated with P53 mutation and patient survival time. Inhibitors of this pathway slow tumor growth and stroma formation, alter immune cell infiltration, and prolong survival of mice. Transcript profiling: ArrayExpress accession number: E-MTAB-3278.