RESUMO
Senescence is a form of cell cycle arrest induced by stress such as DNA damage and oncogenes. However, while arrested, senescent cells secrete a variety of proteins collectively known as the senescence-associated secretory phenotype (SASP), which can reinforce the arrest and induce senescence in a paracrine manner. However, the SASP has also been shown to favor embryonic development, wound healing, and even tumor growth, suggesting more complex physiological roles than currently understood. Here we uncover timely new functions of the SASP in promoting a proregenerative response through the induction of cell plasticity and stemness. We show that primary mouse keratinocytes transiently exposed to the SASP exhibit increased expression of stem cell markers and regenerative capacity in vivo. However, prolonged exposure to the SASP causes a subsequent cell-intrinsic senescence arrest to counter the continued regenerative stimuli. Finally, by inducing senescence in single cells in vivo in the liver, we demonstrate that this activates tissue-specific expression of stem cell markers. Together, this work uncovers a primary and beneficial role for the SASP in promoting cell plasticity and tissue regeneration and introduces the concept that transient therapeutic delivery of senescent cells could be harnessed to drive tissue regeneration.
Assuntos
Plasticidade Celular/fisiologia , Senescência Celular/fisiologia , Regeneração/fisiologia , Via Secretória/fisiologia , Animais , Biomarcadores/metabolismo , Plasticidade Celular/genética , Células Cultivadas , Senescência Celular/genética , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Feminino , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/genética , Queratinócitos/citologia , Queratinócitos/fisiologia , Fígado/citologia , Fígado/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Fenótipo , Regeneração/genética , Via Secretória/genética , Células-Tronco/metabolismoRESUMO
Experimental in vitro models that capture pathophysiological characteristics of human tumours are essential for basic and translational cancer biology. Here, we describe a fully synthetic hydrogel extracellular matrix designed to elicit key phenotypic traits of the pancreatic environment in culture. To enable the growth of normal and cancerous pancreatic organoids from genetically engineered murine models and human patients, essential adhesive cues were empirically defined and replicated in the hydrogel scaffold, revealing a functional role of laminin-integrin α3/α6 signalling in establishment and survival of pancreatic organoids. Altered tissue stiffness-a hallmark of pancreatic cancer-was recapitulated in culture by adjusting the hydrogel properties to engage mechano-sensing pathways and alter organoid growth. Pancreatic stromal cells were readily incorporated into the hydrogels and replicated phenotypic traits characteristic of the tumour environment in vivo. This model therefore recapitulates a pathologically remodelled tumour microenvironment for studies of normal and pancreatic cancer cells in vitro.
Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Adenocarcinoma/metabolismo , Animais , Matriz Extracelular , Humanos , Hidrogéis/metabolismo , Camundongos , Organoides , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Microambiente TumoralRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease and cytotoxic chemotherapy is the standard of care treatment for patients with advanced disease. Here, we investigate how the microenvironment in PDAC liver metastases reacts to chemotherapy and its role in metastatic disease progression post-treatment, an area which is poorly understood. DESIGN: The impact of chemotherapy on metastatic disease progression and immune cell infiltrates was characterised using flow and mass cytometry combined with transcriptional and histopathological analysis in experimental PDAC liver metastases mouse models. Findings were validated in patient derived liver metastases and in an autochthonous PDAC mouse model. Human and murine primary cell cocultures and ex vivo patient-derived liver explants were deployed to gain mechanistical insights on whether and how chemotherapy affects the metastatic tumour microenvironment. RESULTS: We show that in vivo, chemotherapy induces an initial infiltration of proinflammatory macrophages into the liver and activates cytotoxic T cells, leading only to a temporary restraining of metastatic disease progression. However, after stopping treatment, neutrophils are recruited to the metastatic liver via CXCL1 and 2 secretion by metastatic tumour cells. These neutrophils express growth arrest specific 6 (Gas6) which leads to AXL receptor activation on tumour cells enabling their regrowth. Disruption of neutrophil infiltration or inhibition of the Gas6/AXL signalling axis in combination with chemotherapy inhibits metastatic growth. Chemotherapy increases Gas6 expression in circulating neutrophils from patients with metastatic pancreatic cancer and recombinant Gas6 is sufficient to promote tumour cell proliferation ex vivo, in patient-derived metastatic liver explants. CONCLUSION: Combining chemotherapy with Gas6/AXL or neutrophil targeted therapy could provide a therapeutic benefit for patients with metastatic pancreatic cancer.
Assuntos
Antineoplásicos , Carcinoma Ductal Pancreático , Neoplasias Hepáticas , Neoplasias Pancreáticas , Animais , Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/patologia , Progressão da Doença , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Camundongos , Metástase Neoplásica , Neutrófilos/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
An ever-increasing array of imaging technologies are being used in the study of complex biological samples, each of which provides complementary, occasionally overlapping information at different length scales and spatial resolutions. It is important to understand the information provided by one technique in the context of the other to achieve a more holistic overview of such complex samples. One way to achieve this is to use annotations from one modality to investigate additional modalities. For microscopy-based techniques, these annotations could be manually generated using digital pathology software or automatically generated by machine learning (including deep learning) methods. Here, we present a generic method for using annotations from one microscopy modality to extract information from complementary modalities. We also present a fast, general, multimodal registration workflow [evaluated on multiple mass spectrometry imaging (MSI) modalities, matrix-assisted laser desorption/ionization, desorption electrospray ionization, and rapid evaporative ionization mass spectrometry] for automatic alignment of complex data sets, demonstrating an order of magnitude speed-up compared to previously published work. To demonstrate the power of the annotation transfer and multimodal registration workflows, we combine MSI, histological staining (such as hematoxylin and eosin), and deep learning (automatic annotation of histology images) to investigate a pancreatic cancer mouse model. Neoplastic pancreatic tissue regions, which were histologically indistinguishable from one another, were observed to be metabolically different. We demonstrate the use of the proposed methods to better understand tumor heterogeneity and the tumor microenvironment by transferring machine learning results freely between the two modalities.
Assuntos
Aprendizado Profundo , Animais , Técnicas Histológicas , Camundongos , Imagem Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fluxo de TrabalhoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 4% and desperately needs novel effective therapeutics. Integrin αvß6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed that high levels of ß6 mRNA correlated strongly with significantly poorer survival (n = 491 cases, p = 3.17 × 10-8 ). In two separate cohorts, we showed that over 80% of PDACs expressed αvß6 protein and that paired metastases retained αvß6 expression. In vitro, integrin αvß6 promoted PDAC cell growth, survival, migration, and invasion. Treatment of both αvß6-positive human PDAC xenografts and transgenic mice bearing αvß6-positive PDAC with the αvß6 blocking antibody 264RAD, combined with gemcitabine, significantly reduced tumour growth (p < 0.0001) and increased survival (log-rank test, p < 0.05). Antibody therapy was associated with suppression of tumour cell activity (suppression of pErk growth signals, increased apoptosis seen as activated caspase-3) and suppression of the pro-tumourigenic microenvironment (suppression of TGFß signalling, fewer αSMA-positive myofibroblasts, decreased blood vessel density). These data show that αvß6 promotes PDAC growth through both tumour cell and tumour microenvironment mechanisms and represents a valuable target for PDAC therapy. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Antígenos de Neoplasias/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Integrinas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Antígenos de Neoplasias/genética , Antineoplásicos Imunológicos/farmacologia , Apoptose , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/secundário , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Fosfatase 6 de Especificidade Dupla/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes ras , Humanos , Integrases/genética , Integrinas/antagonistas & inibidores , Integrinas/genética , Itália , Camundongos Nus , Camundongos Transgênicos , Invasividade Neoplásica , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Carga Tumoral , Microambiente Tumoral , Reino Unido , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is among the most deadly solid tumours. This is due to a generally late-stage diagnosis of a primarily treatment-refractory disease. Several large-scale sequencing and mass spectrometry approaches have identified key drivers of this disease and in doing so highlighted the vast heterogeneity of lower frequency mutations that make clinical trials of targeted agents in unselected patients increasingly futile. There is a clear need for improved biomarkers to guide effective targeted therapies, with biomarker-driven clinical trials for personalised medicine becoming increasingly common in several cancers. Interestingly, many of the aberrant signalling pathways in PDAC rely on downstream signal transduction through the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways, which has led to the development of several approaches to target these key regulators, primarily as combination therapies. The following review discusses the trend of PDAC therapy towards molecular subtyping for biomarker-driven personalised therapies, highlighting the key pathways under investigation and their relationship to the PI3K pathway.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Medicina de Precisão , Inibidores de Proteínas Quinases/uso terapêutico , Biomarcadores Tumorais/análise , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Progressão da Doença , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Transdução de SinaisRESUMO
Pancreatic cancer is accompanied by a fibrotic reaction that alters interactions between tumor cells and the stroma to promote tumor progression. Consequently, strategies to target the tumor stroma might be used to treat patients with pancreatic cancer. We review recently developed approaches for reshaping the pancreatic tumor stroma and discuss how these might improve patient outcomes. We also describe relationships between the pancreatic tumor extracellular matrix, the vasculature, the immune system, and metabolism, and discuss the implications for the development of stromal compartment-specific therapies.
Assuntos
Antineoplásicos/uso terapêutico , Matriz Extracelular/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Células Estromais/efeitos dos fármacos , Microambiente Tumoral , Animais , Biomarcadores Tumorais/metabolismo , Comunicação Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Células Estromais/imunologia , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
Macroautophagy (hereafter referred to as autophagy) is a process in which organelles termed autophagosomes deliver cytoplasmic constituents to lysosomes for degradation. Autophagy has a major role in cellular homeostasis and has been implicated in various forms of human disease. The role of autophagy in cancer seems to be complex, with reports indicating both pro-tumorigenic and tumour-suppressive roles. Here we show, in a humanized genetically-modified mouse model of pancreatic ductal adenocarcinoma (PDAC), that autophagy's role in tumour development is intrinsically connected to the status of the tumour suppressor p53. Mice with pancreases containing an activated oncogenic allele of Kras (also called Ki-Ras)--the most common mutational event in PDAC--develop a small number of pre-cancerous lesions that stochastically develop into PDAC over time. However, mice also lacking the essential autophagy genes Atg5 or Atg7 accumulate low-grade, pre-malignant pancreatic intraepithelial neoplasia lesions, but progression to high-grade pancreatic intraepithelial neoplasias and PDAC is blocked. In marked contrast, in mice containing oncogenic Kras and lacking p53, loss of autophagy no longer blocks tumour progression, but actually accelerates tumour onset, with metabolic analysis revealing enhanced glucose uptake and enrichment of anabolic pathways, which can fuel tumour growth. These findings provide considerable insight into the role of autophagy in cancer and have important implications for autophagy inhibition in cancer therapy. In this regard, we also show that treatment of mice with the autophagy inhibitor hydroxychloroquine, which is currently being used in several clinical trials, significantly accelerates tumour formation in mice containing oncogenic Kras but lacking p53.
Assuntos
Autofagia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Genes p53/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteína Supressora de Tumor p53/genética , Alelos , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteína 5 Relacionada à Autofagia , Proteína 7 Relacionada à Autofagia , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Glucose/metabolismo , Glicólise/genética , Humanos , Hidroxicloroquina/farmacologia , Metabolômica , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/genética , Proteína Oncogênica p21(ras)/genética , Neoplasias Pancreáticas/metabolismo , Via de Pentose Fosfato/genética , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Análise de Sobrevida , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mutations in both RAS and the PTEN/PIK3CA/AKT signaling module are found in the same human tumors. PIK3CA and AKT are downstream effectors of RAS, and the selective advantage conferred by mutation of two genes in the same pathway is unclear. Based on a comparative molecular analysis, we show that activated PIK3CA/AKT is a weaker inducer of senescence than is activated RAS. Moreover, concurrent activation of RAS and PIK3CA/AKT impairs RAS-induced senescence. In vivo, bypass of RAS-induced senescence by activated PIK3CA/AKT correlates with accelerated tumorigenesis. Thus, not all oncogenes are equally potent inducers of senescence, and, paradoxically, a weak inducer of senescence (PIK3CA/AKT) can be dominant over a strong inducer of senescence (RAS). For tumor growth, one selective advantage of concurrent mutation of RAS and PTEN/PIK3CA/AKT is suppression of RAS-induced senescence. Evidence is presented that this new understanding can be exploited in rational development and targeted application of prosenescence cancer therapies.
Assuntos
Genes ras , Neoplasias/enzimologia , Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Idoso , Animais , Linhagem Celular Transformada , Proliferação de Células , Senescência Celular/genética , Senescência Celular/fisiologia , Classe I de Fosfatidilinositol 3-Quinases , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Neoplasias/patologia , PTEN Fosfo-Hidrolase/antagonistas & inibidores , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de SinaisRESUMO
The prognosis of cholangiocarcinoma (CC) is dismal. Notch has been identified as a potential driver; forced exogenous overexpression of Notch1 in hepatocytes results in the formation of biliary tumors. In human disease, however, it is unknown which components of the endogenously signaling pathway are required for tumorigenesis, how these orchestrate cancer, and how they can be targeted for therapy. Here we characterize Notch in human-resected CC, a toxin-driven model in rats, and a transgenic mouse model in which p53 deletion is targeted to biliary epithelia and CC induced using the hepatocarcinogen thioacetamide. We find that across species, the atypical receptor NOTCH3 is differentially overexpressed; it is progressively up-regulated with disease development and promotes tumor cell survival via activation of PI3k-Akt. We use genetic KO studies to show that tumor growth significantly attenuates after Notch3 deletion and demonstrate signaling occurs via a noncanonical pathway independent of the mediator of classical Notch, Recombinant Signal Binding Protein for Immunoglobulin Kappa J Region (RBPJ). These data present an opportunity in this aggressive cancer to selectively target Notch, bypassing toxicities known to be RBPJ dependent.
Assuntos
Carcinogênese/genética , Colangiocarcinoma/genética , Neoplasias Experimentais/genética , Prognóstico , Receptor Notch3/genética , Animais , Colangiocarcinoma/patologia , Humanos , Região de Junção de Imunoglobulinas/genética , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais/patologia , Fosfatidilinositol 3-Quinases/genética , Ratos , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
OBJECTIVE: Extensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4. DESIGN: Sensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens). RESULTS: Subtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach. CONCLUSION: This study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular/métodos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação , Piperazinas/uso terapêutico , Prognóstico , Piridinas/uso terapêutico , Proteína do Retinoblastoma/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatitis is a significant clinical problem and the lack of effective therapeutic options means that treatment is often palliative rather than curative. A deeper understanding of the pathogenesis of both acute and chronic pancreatitis is necessary to develop new therapies. Pathological changes in pancreatitis are dependent on innate immune cell recruitment to the site of initial tissue damage, and on the coordination of downstream inflammatory pathways. The chemokine receptor CXCR2 drives neutrophil recruitment during inflammation, and to investigate its role in pancreatic inflammation, we induced acute and chronic pancreatitis in wild-type and Cxcr2(-/-) mice. Strikingly, Cxcr2(-/-) mice were strongly protected from tissue damage in models of acute pancreatitis, and this could be recapitulated by neutrophil depletion or by the specific deletion of Cxcr2 from myeloid cells. The pancreata of Cxcr2(-/-) mice were also substantially protected from damage during chronic pancreatitis. Neutrophil depletion was less effective in this model, suggesting that CXCR2 on non-neutrophils contributes to the development of chronic pancreatitis. Importantly, pharmacological inhibition of CXCR2 in wild-type mice replicated the protection seen in Cxcr2(-/-) mice in acute and chronic models of pancreatitis. Moreover, acute pancreatic inflammation was reversible by inhibition of CXCR2. Thus, CXCR2 is critically involved in the development of acute and chronic pancreatitis in mice, and its inhibition or loss protects against pancreatic damage. CXCR2 may therefore be a viable therapeutic target in the treatment of pancreatitis.
Assuntos
Anti-Inflamatórios/farmacologia , Pâncreas/efeitos dos fármacos , Pancreatite Crônica/prevenção & controle , Pancreatite/prevenção & controle , Peptídeos/farmacologia , Receptores de Interleucina-8B/antagonistas & inibidores , Doença Aguda , Animais , Ceruletídeo , Citoproteção , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Pâncreas/imunologia , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/genética , Pancreatite/imunologia , Pancreatite/metabolismo , Pancreatite/patologia , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/genética , Pancreatite Crônica/imunologia , Pancreatite Crônica/metabolismo , Pancreatite Crônica/patologia , Receptores de Interleucina-8B/deficiência , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/imunologia , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is often lethal because it is highly invasive and metastasizes rapidly. The actin-bundling protein fascin has been identified as a biomarker of invasive and advanced PDAC and regulates cell migration and invasion in vitro. We investigated fascin expression and its role in PDAC progression in mice. METHODS: We used KRas(G12D) p53(R172H) Pdx1-Cre (KPC) mice to investigate the effects of fascin deficiency on development of pancreatic intraepithelial neoplasia (PanIn), PDAC, and metastasis. We measured levels of fascin in PDAC cell lines and 122 human resected PDAC samples, along with normal ductal and acinar tissues; we associated levels with patient outcomes. RESULTS: Pancreatic ducts and acini from control mice and early-stage PanINs from KPC mice were negative for fascin, but approximately 6% of PanIN3 and 100% of PDAC expressed fascin. Fascin-deficient KRas(G12D) p53(R172H) Pdx1-Cre mice had longer survival times, delayed onset of PDAC, and a lower PDAC tumor burdens than KPC mice; loss of fascin did not affect invasion of PDAC into bowel or peritoneum in mice. Levels of slug and fascin correlated in PDAC cells; slug was found to regulate transcription of Fascin along with the epithelial-mesenchymal transition. In PDAC cell lines and cells from mice, fascin concentrated in filopodia and was required for their assembly and turnover. Fascin promoted intercalation of filopodia into mesothelial cell layers and cell invasion. Nearly all human PDAC samples expressed fascin, and higher fascin histoscores correlated with poor outcomes, vascular invasion, and time to recurrence. CONCLUSIONS: The actin-bundling protein fascin is regulated by slug and involved in late-stage PanIN and PDAC formation in mice. Fascin appears to promote formation of filopodia and invasive activities of PDAC cells. Its levels in human PDAC correlate with outcomes and time to recurrence, indicating it might be a marker or therapeutic target for pancreatic cancer.
Assuntos
Carcinoma in Situ/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Carcinoma in Situ/genética , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/secundário , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Transição Epitelial-Mesenquimal , Humanos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Invasividade Neoplásica , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Prognóstico , Pseudópodes/metabolismo , Interferência de RNA , Fatores de Transcrição da Família Snail , Análise de Sobrevida , Fatores de Tempo , Fatores de Transcrição/genética , TransfecçãoRESUMO
OBJECTIVE: Pancreatic cancer is a leading cause of cancer-related death in the Western world. Current chemotherapy regimens have modest survival benefit. Thus, novel, effective therapies are required for treatment of this disease. DESIGN: Activating KRAS mutation almost always drives pancreatic tumour initiation, however, deregulation of other potentially druggable pathways promotes tumour progression. PTEN loss leads to acceleration of Kras(G12D)-driven pancreatic ductal adenocarcinoma (PDAC) in mice and these tumours have high levels of mammalian target of rapamycin (mTOR) signalling. To test whether these KRAS PTEN pancreatic tumours show mTOR dependence, we compared response to mTOR inhibition in this model, to the response in another established model of pancreatic cancer, KRAS P53. We also assessed whether there was a subset of pancreatic cancer patients who may respond to mTOR inhibition. RESULTS: We found that tumours in KRAS PTEN mice exhibit a remarkable dependence on mTOR signalling. In these tumours, mTOR inhibition leads to proliferative arrest and even tumour regression. Further, we could measure response using clinically applicable positron emission tomography imaging. Importantly, pancreatic tumours driven by activated KRAS and mutant p53 did not respond to treatment. In human tumours, approximately 20% of cases demonstrated low PTEN expression and a gene expression signature that overlaps with murine KRAS PTEN tumours. CONCLUSIONS: KRAS PTEN tumours are uniquely responsive to mTOR inhibition. Targeted anti-mTOR therapies may offer clinical benefit in subsets of human PDAC selected based on genotype, that are dependent on mTOR signalling. Thus, the genetic signatures of human tumours could be used to direct pancreatic cancer treatment in the future.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/antagonistas & inibidores , Carcinoma Ductal Pancreático/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Esquema de Medicação , Regulação Neoplásica da Expressão Gênica , Humanos , Injeções Intraperitoneais , Camundongos , Camundongos Mutantes , Mutação , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Tomografia por Emissão de Pósitrons , Proteínas Proto-Oncogênicas p21(ras)/deficiência , Proteínas Proto-Oncogênicas p21(ras)/genética , Serina-Treonina Quinases TOR/metabolismo , Resultado do Tratamento , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Basic in vitro systems can be used to model and assess complex diseases, such as cancer. Recent advances in this field include the incorporation of multiple cell types and extracellular matrix proteins into three-dimensional (3D) models to recapitulate the structure, organization and functionality of live tissue in situ. Cells within such a 3D environment behave very differently from cells on two-dimensional (2D) substrates, as cell-matrix interactions trigger signalling pathways and cellular responses in 3D, which may not be observed in 2D. Thus, the use of 3D systems can be advantageous for the assessment of disease progression over 2D set-ups alone. Here, we highlight the current advantages and challenges of employing 3D systems in the study of cancer and provide an overview to guide the appropriate use of distinct models in cancer research.
Assuntos
Comunicação Celular , Modelos Biológicos , Neoplasias/patologia , Microambiente Tumoral , Animais , Técnicas de Cocultura , Humanos , Transdução de SinaisRESUMO
Src family kinase activity is elevated in a number of human cancers including breast cancer. This increased activity has been associated with aggressive disease and poor prognosis. Src inhibitors are currently in clinical development with a number of trials currently assessing their activity in breast cancer. However, the results to date have been disappointing and a further evaluation of the preclinical effects of Src inhibitors is required to help establish whether these agents will be useful in the treatment of breast cancer. In this study we investigate the effects of dasatinib, which is a potent inhibitor of Src family kinases, on the initiation and development of breast cancer in a genetically engineered model of the disease. The mouse model utilized is driven by expression of activated ErbB-2 under the transcriptional control of its endogenous promoter coupled with conditional loss of Pten under the control of Cre recombinase expressed by the BLG promoter. We show that daily oral administration of dasatinib delays tumour onset and increases overall survival but does not inhibit the proliferation of established tumours. The striking difference between the dasatinib-treated group of tumours and the vehicle controls was the prominent squamous metaplasia that was seen in six out of 11 dasatinib-treated tumours. This was accompanied by a dramatic up-regulation of both E-cadherin and ß-catenin and down-regulation of ErbB-2 in the dasatinib-treated tumours. Dasatinib also inhibited both the migration and the invasion of tumour-derived cell lines in vitro. Together these data support the argument that benefits of Src inhibitors may predominate in early or even pre-invasive disease.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/prevenção & controle , Neoplasias Mamárias Experimentais/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Quinases da Família src/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dasatinibe , Relação Dose-Resposta a Droga , Esquema de Medicação , Elementos Facilitadores Genéticos , Feminino , Regulação Neoplásica da Expressão Gênica , Genes erbB-2 , Integrases/genética , Integrases/metabolismo , Lactoglobulinas/genética , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Invasividade Neoplásica , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Tiazóis/administração & dosagem , Fatores de Tempo , beta Catenina/genética , beta Catenina/metabolismo , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Pancreatico-biliary adenocarcinomas (PBA) have a poor prognosis. Diagnosis is usually achieved by imaging and/or endoscopy with confirmatory cytology. Cytological interpretation can be difficult especially in the setting of chronic pancreatitis/cholangitis. Immunohistochemistry (IHC) biomarkers could act as an adjunct to cytology to improve the diagnosis. Thus, we performed a meta-analysis and selected KOC, S100P, mesothelin and MUC1 for further validation in PBA resection specimens. METHODS: Tissue microarrays containing tumour and normal cores in a ratio of 3:2, from 99 surgically resected PBA patients, were used for IHC. IHC was performed on an automated platform using antibodies against KOC, S100P, mesothelin and MUC1. Tissue cores were scored for staining intensity and proportion of tissue stained using a Histoscore method (range, 0-300). Sensitivity and specificity for individual biomarkers, as well as biomarker panels, were determined with different cut-offs for positivity and compared by summary receiver operating characteristic (ROC) curve. RESULTS: The expression of all four biomarkers was high in PBA versus normal ducts, with a mean Histoscore of 150 vs. 0.4 for KOC, 165 vs. 0.3 for S100P, 115 vs. 0.5 for mesothelin and 200 vs. 14 for MUC1 (p < .0001 for all comparisons). Five cut-offs were carefully chosen for sensitivity/specificity analysis. Four of these cut-offs, namely 5%, 10% or 20% positive cells and Histoscore 20 were identified using ROC curve analysis and the fifth cut-off was moderate-strong staining intensity. Using 20% positive cells as a cut-off achieved higher sensitivity/specificity values: KOC 84%/100%; S100P 83%/100%; mesothelin 88%/92%; and MUC1 89%/63%. Analysis of a panel of KOC, S100P and mesothelin achieved 100% sensitivity and 99% specificity if at least 2 biomarkers were positive for 10% cut-off; and 100% sensitivity and specificity for 20% cut-off. CONCLUSION: A biomarker panel of KOC, S100P and mesothelin with at least 2 biomarkers positive was found to be an optimum panel with both 10% and 20% cut-offs in resection specimens from patients with PBA.
RESUMO
Pancreatic ductal adenocarcinoma is an aggressive malignancy with limited treatment options. Chemotherapy offers little benefit and, although there is some evidence that radiotherapy may improve response, its use in the clinical management of pancreatic cancer remains controversial due to conflicting reports on its survival benefit. There has also been a lack of clinical trials that directly investigate the efficacy of radiotherapy in pancreatic cancer. The limited progress in the development of radiotherapeutic strategies in pancreatic cancer can be attributed, at least in part, to a dearth of preclinical research and our limited understanding of the effects of radiation on the pancreatic tumour microenvironment. In this Perspective, we discuss how insight into the immunosuppressive tumour microenvironment and the complex signalling between tumour and stromal cells following radiation is needed to develop effective radiosensitising strategies for pancreatic cancer. We also highlight that to have the best chance for successful clinical translation, more preclinical research is required in appropriately complex models.
Assuntos
Neoplasias Pancreáticas , Microambiente Tumoral , Animais , Humanos , Neoplasias Pancreáticas/radioterapia , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Microambiente Tumoral/efeitos da radiaçãoRESUMO
The value of radiotherapy in the treatment of pancreatic cancer has been the subject of much debate but limited preclinical research. We hypothesise that the poor translation of radiation research into clinical trials of radiotherapy in pancreatic cancer is due, in part, to inadequate preclinical study models. Here, we developed and refined methods for targeted irradiation in autochthonous mouse models of pancreatic cancer, using a small animal radiotherapy research platform. We tested and optimised strategies for administration of contrast agents, iohexol and the liver imaging agent Fenestra LC, to enable the use of computed tomography imaging in tumour localisation. We demonstrate accurate tumour targeting, negligible off-target effects and therapeutic efficacy, depending on dose, number of fractions and tumour size, and provide a proof of concept that precise radiation can be delivered effectively to mouse pancreatic tumours with a clinically relevant microenvironment. This advance will allow investigation of the radiation response in murine pancreatic cancer, discovery of mechanisms and biomarkers of radiosensitivity or resistance, and development of radiosensitising strategies to inform clinical trials for precision radiotherapy in this disease.