RESUMEN
Survival from pancreatic cancer is poor because most cancers are diagnosed in the late stages and there are no therapies to prevent the progression of precancerous pancreatic intraepithelial neoplasms (PanINs). Inhibiting mutant KRASG12D, the primary driver mutation in most human pancreatic cancers, has been challenging. The cholecystokinin-B receptor (CCK-BR) is absent in the normal pancreas but becomes expressed in high grade PanIN lesions and is over-expressed in pancreatic cancer making it a prime target for therapy. We developed a biodegradable nanoparticle polyplex (NP) that binds selectively to the CCK-BR on PanINs and pancreatic cancer to deliver gene therapy. PanIN progression was halted and the pancreas extracellular matrix rendered less carcinogenic in P48-Cre/LSL-KrasG12D/+ mice treated with the CCK-BR targeted NP loaded with siRNA to mutant Kras. The targeted NP also slowed proliferation, decreased metastases and improved survival in mice bearing large orthotopic pancreatic tumors. Safety and toxicity studies were performed in immune competent mice after short or long-term exposure and showed no off-target toxicity by histological or biochemical evaluation. Precision therapy with target-specific NPs provides a novel approach to slow progression of advanced pancreatic cancer and also prevents the development of pancreatic cancer in high-risk subjects without toxicity to other tissues.
Asunto(s)
Carcinoma in Situ , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ratones , Humanos , Animales , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Modelos Animales de Enfermedad , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/prevención & control , Páncreas/metabolismo , Carcinogénesis/genética , Carcinogénesis/patología , Carcinoma in Situ/genética , Carcinoma Ductal Pancreático/patología , Neoplasias PancreáticasRESUMEN
Nonalcoholic steatohepatitis (NASH) is associated with obesity, metabolic syndrome, and dysbiosis of the gut microbiome. Cholecystokinin (CCK) is released by saturated fats and plays an important role in bile acid secretion. CCK receptors are expressed on cholangiocytes, and CCK-B receptor expression increases in the livers of mice with NASH. The farnesoid X receptor (FXR) is involved in bile acid transport and is a target for novel therapeutics for NASH. The aim of this study was to examine the role of proglumide, a CCK receptor inhibitor, in a murine model of NASH and its interaction at FXR. Mice were fed a choline deficient ethionine (CDE) diet to induce NASH. Some CDE-fed mice received proglumide-treated drinking water. Blood was collected and liver tissues were examined histologically. Proglumide's interaction at FXR was evaluated by computer modeling, a luciferase reporter assay, and tissue FXR expression. Stool microbiome was analyzed by RNA-Sequencing. CDE-fed mice developed NASH and the effect was prevented by proglumide. Computer modeling demonstrated specific binding of proglumide to FXR. Proglumide binding in the reporter assay was consistent with a partial agonist at the FXR with a mean binding affinity of 215 nM. FXR expression was significantly decreased in livers of CDE-fed mice compared to control livers, and proglumide restored FXR expression to normal levels. Proglumide therapy altered the microbiome signature by increasing beneficial and decreasing harmful bacteria. These data highlight the potential novel mechanisms by which proglumide therapy may improve NASH through interaction with the FXR and consequent alteration of the gut microbiome.
Asunto(s)
Bacterias/clasificación , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Proglumida/administración & dosificación , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Bacterias/genética , Bacterias/aislamiento & purificación , Modelos Animales de Enfermedad , Microbioma Gastrointestinal/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Filogenia , Proglumida/química , Proglumida/farmacología , Receptores Citoplasmáticos y Nucleares/químicaRESUMEN
BACKGROUND AND AIMS: Chronic pancreatitis is associated with recurrent inflammation, pain, fibrosis, and loss of exocrine and endocrine pancreatic function and risk of cancer. We hypothesized that activation of the CCK receptor contributes to pancreatitis and blockade of this pathway would improve chronic pancreatitis. METHODS: Two murine models were used to determine whether CCK receptor blockade with proglumide could prevent and reverse histologic and biochemical features of chronic pancreatitis: the 6-week repetitive chronic cerulein injection model and the modified 75% choline-deficient ethionine (CDE) diet. In the CDE-fed model, half the mice received water supplemented with proglumide, for 18 weeks. After chronic pancreatitis was established in the cerulein model, half the mice were treated with proglumide and half with water. Histology was scored in a blinded fashion for inflammation, fibrosis and acinar ductal metaplasia (ADM) and serum lipase levels were measured. RNA was extracted and examined for differentially expressed fibrosis genes. RESULTS: Proglumide therapy decreased pancreatic weight in the CDE diet study and the cerulein-induced chronic pancreatitis model. Fibrosis, inflammation, and ADM scores were significantly reduced in both models. Lipase values improved with proglumide but not in controls in both models. Proglumide decreased pancreas mRNA expression of amylase, collagen-4, and TGFßR2 gene expression by 44, 38, and 25%, respectively, compared to control mice. CONCLUSION: New strategies are needed to decreased inflammation and reduce fibrosis in chronic pancreatitis. CCK receptor antagonist therapy may improve chronic pancreatitis by reversing fibrosis and inflammation. The decrease in ADM may reduce the risk of the development of pancreatic cancer.
Asunto(s)
Páncreas/patología , Pancreatitis Crónica/tratamiento farmacológico , Proglumida/farmacología , Receptores de Colecistoquinina/agonistas , Animales , Ceruletida , Enfermedad Crónica , Modelos Animales de Enfermedad , Fibrosis , Inflamación , Lipasa/sangre , Ratones , Pancreatitis Crónica/inducido químicamente , Pancreatitis Crónica/patologíaRESUMEN
BACKGROUND AND AIMS: Nonalcoholic steatohepatitis (NASH) is a common inflammatory liver condition that may lead to cirrhosis and hepatocellular carcinoma (HCC). Risk factors for NASH include a saturated fat diet, altered lipid metabolism, and genetic and epigenetic factors, including microRNAs. Serum levels of cholecystokinin (CCK) are elevated in mice and humans that consume a high-saturated fat diet. CCK receptors (CCK-Rs) have been reported on fibroblasts which when activated can induce fibrosis; however, their role in hepatic fibrosis remains unknown. We hypothesized that elevated levels of CCK acting on the CCK-Rs play a role in the development of NASH and in NASH-associated HCC. METHODS: We performed a NASH Prevention study and Reversal study in mice fed a saturated fat 75% choline-deficient-ethionine-supplemented (CDE) diet for 12 or 18 weeks. In each study, half of the mice received untreated drinking water, while the other half received water supplemented with the CCK-R antagonist proglumide. CCK-R expression was evaluated in mouse liver and murine HCC cells. RESULTS: CCK receptor antagonist treatment not only prevented NASH but also reversed hepatic inflammation, fibrosis, and steatosis and normalized hepatic transaminases after NASH was established. Thirty-five percent of the mice on the CDE diet developed HCC compared with none in the proglumide-treated group. We found that CCK-BR expression was markedly upregulated in mouse CDE liver and HCC cells compared with normal hepatic parenchymal cells, and this expression was epigenetically regulated by microRNA-148a. CONCLUSION: These results support the novel role of CCK receptors in the pathogenesis of NASH and HCC.
Asunto(s)
Carcinoma Hepatocelular/prevención & control , Antagonistas de Hormonas/farmacología , Neoplasias Hepáticas/prevención & control , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Proglumida/farmacología , Receptor de Colecistoquinina B/antagonistas & inhibidores , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Deficiencia de Colina/complicaciones , Modelos Animales de Enfermedad , Epigénesis Genética , Etionina , Femenino , Regulación Neoplásica de la Expresión Génica , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Receptor de Colecistoquinina B/genética , Receptor de Colecistoquinina B/metabolismo , Transducción de SeñalRESUMEN
Growth of pancreatic cancer is stimulated by gastrin in both a paracrine and an autocrine fashion. Traditional therapies have not significantly improved survival, and recently pancreatic cancer has been deemed a "cold" tumor due to its poor response to immunotherapy. Strategies to improve survival of pancreatic cancer are desperately needed. In the current investigation, we studied the effects of an anti-gastrin cancer vaccine, polyclonal antibody stimulator (PAS; formerly called G17DT and Gastrimmune), used alone or in combination with a programmed cell death receptor (PD)-1 immune checkpoint antibody on pancreatic cancer growth, metastases, and the tumor microenvironment (TME). Immune-competent female C57BL/6 mice bearing syngeneic orthotopic murine pancreatic cancer treated with PAS had significantly smaller tumors and fewer metastases. Examination of the TME demonstrated decreased fibrosis with fewer M2 and more M1 tumor-associated macrophages. Expression of the E-cadherin gene was significantly increased and expression of the TGFßR2 gene was decreased compared with controls. Mice treated with PAS or the combination of PAS and PD-1 antibody exhibited significantly less tumor expression of phospho-paxillin, the focal adhesion protein ß-catenin, and matrix metalloproteinase-7. This study suggests that inhibition of the cancer-promoting effects of gastrin in pancreatic cancer can decrease metastases by altering the TME and decreasing pathways that activate the epithelial mesenchymal transition. The PAS vaccine appears to change the TME, making it more susceptible to therapy with an immune checkpoint antibody. This novel combination of two immunotherapies may improve survival of pancreatic cancer by decreasing both tumor growth and metastasis formation.NEW & NOTEWORTHY Survival from advanced pancreatic cancer is poor, in part due to dense fibrosis of the tumor microenvironment, increased number of M2-polarized macrophages that promote angiogenesis and invasion, and lack of "target-specific" therapy. Herein, we report that a tumor vaccine that selectively targets gastrin decreases pancreatic cancer growth and metastases. Furthermore, the gastrin vaccine polyclonal antibody stimulator alters the tumor microenvironment rendering it more responsive to immunotherapy with a programmed cell death receptor-1 immune checkpoint antibody.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Gastrinas/inmunología , Inmunoterapia/métodos , Neoplasias Pancreáticas/terapia , Animales , Cadherinas/genética , Cadherinas/metabolismo , Vacunas contra el Cáncer/uso terapéutico , Línea Celular , Línea Celular Tumoral , Femenino , Gastrinas/uso terapéutico , Macrófagos/metabolismo , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neoplasias Pancreáticas/patología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Microambiente Tumoral , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
The gastrointestinal peptide cholecystokinin (CCK) is released from the duodenum in response to dietary fat to aid in digestion, and plasma CCK levels are elevated with the consumption of high-fat diets. CCK is also a trophic peptide for the pancreas and has also been shown to stimulate growth of pancreatic cancer. In the current investigation, we studied the influence of a diet high in saturated fat on the growth of pancreatic cancer in syngeneic murine models before the mice became obese to exclude the confounding factors associated with obesity. The high-fat diet significantly increased growth and metastasis of pancreatic cancer compared with the control diet, and the stimulatory effect was blocked by the CCK-receptor antagonist proglumide. We then selectively knocked out the CCK receptor on the pancreatic cancer cells using clustered regularly interspaced short palindromic repeats technology and showed that without CCK-receptors, dietary fat was unable to stimulate cancer growth. We next demonstrated that dietary fat failed to influence pancreatic cancer xenograft growth in genetically engineered CCK peptide knockout mice. The tumor-associated fibrosis that is so prevalent in the pancreatic cancer microenvironment was significantly decreased with CCK-receptor antagonist therapy because fibroblasts also have CCK receptors. The CCK-receptor antagonist proglumide also altered tumor metalloprotease expression and increased tumor suppressor genes by a PCR array. Our studies confirm that a diet high in saturated fat promotes growth of pancreatic cancer and the action is mediated by the CCK-receptor pathway. NEW & NOTEWORTHY Diets high in long-chain saturated fats promote growth of pancreatic cancer independent of obesity. The mechanism through which dietary fat promotes cancer is mediated through the cholecystokinin (CCK) receptor pathway. Therapy with a CCK-receptor antagonist altered the tumor microenvironment by reducing fibrosis, increasing cluster of differentiation 8+ lymphocytes, increasing tumor suppressor genes, and thus decreasing metastases. Use of CCK-receptor antagonist therapy with standard chemotherapy for pancreatic cancer may improve response by altering the tumor microenvironment.
Asunto(s)
Grasas de la Dieta/efectos adversos , Neoplasias Pancreáticas/etiología , Receptores de Colecistoquinina/metabolismo , Microambiente Tumoral , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Femenino , Fibrosis , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/tratamiento farmacológico , Proglumida/farmacología , Proglumida/uso terapéutico , Receptores de Colecistoquinina/antagonistas & inhibidores , Receptores de Colecistoquinina/genéticaRESUMEN
BACKGROUND & AIMS: Current hepatitis B virus (HBV) management is challenging as treatment with nucleos(t)ide analogues needs to be maintained indefinitely and because interferon (IFN)-α therapy is associated with considerable toxicity. Previously, we showed that linking IFNα to apolipoprotein A-I generates a molecule (IA) with distinct antiviral and immunostimulatory activities which lacks the hematological toxicity of IFNα. METHODS: Here, we analyse the antiviral potential of an adeno-associated vector encoding IFNα fused to apolipoprotein A-I (AAV-IA) in comparison to a vector encoding only IFNα (AAV-IFN) in two animal models of chronic hepadnavirus infection. RESULTS: In HBV transgenic mice, we found that both vectors induced marked reductions in serum and liver HBV DNA and in hepatic HBV RNA but AAV-IFN caused lethal pancytopenia. Woodchucks with chronic hepatitis virus (WHV) infection that were treated by intrahepatic injection of vectors encoding the woodchuck sequences (AAV-wIFN or AAV-wIA), experienced only a slight reduction of viremia which was associated with hematological toxicity and high mortality when using AAV-wIFN, while AAV-wIA was well tolerated. However, when we tested AAV-wIA or a control vector encoding woodchuck apolipoprotein A-I (AAV-wApo) in combination with entecavir, we found that AAV-wApo-treated animals exhibited an immediate rebound of viral load upon entecavir withdrawal while, in AAV-wIA-treated woodchucks, viremia and antigenemia remained at low levels for several weeks following entecavir interruption. CONCLUSIONS: Treatment with AAV-IA is safe and elicits antiviral effects in animal models with difficult to treat chronic hepadnavirus infection. AAV-IA in combination with nucleos(t)ide analogues represents a promising approach for the treatment of HBV infection in highly viremic patients.
Asunto(s)
Apolipoproteína A-I/metabolismo , ADN Viral/genética , Terapia Genética/métodos , Hepadnaviridae/genética , Hepatitis B Crónica/terapia , Interferón-alfa/uso terapéutico , Hígado/efectos de los fármacos , Animales , Antivirales/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos , Hepatitis B Crónica/genética , Hepatitis B Crónica/virología , Hígado/virología , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
OBJECTIVES: To analyze whether use of proton pump inhibitors increase the risk for pancreatic cancer in a mouse model and human clinical cohorts. METHODS: p48-Cre/LSL-KrasG12D mice that develop precancerous pancreatic intraepithelial neoplasia (PanINs) were treated with low- or high-dose proton pump inhibitors (PPIs) orally for 1 and 4 months. The mechanism for the cholecystokinin receptor 2 (CCK-2R) activation was investigated in vitro. Two resources were employed to analyze the risk of pancreatic cancer in human subjects with PPI use. RESULTS: Serum gastrin levels were increased 8-fold (P < 0.0001) in mice treated with chronic high-dose PPIs, and this change correlated with an increase (P = 0.02) in PanIN grade and the development of microinvasive cancer. The CCK-2R expression was regulated by microRNA-148a in the p48-Cre/LSL-KrasG12D mice pancreas and in human pancreatic cancer cells in vitro. Proton pump inhibitor consumption in human subjects was correlated with pancreatic cancer risk (odds ratio, 1.54). A validation analysis conducted using the large-scale United Kingdom Biobank database confirmed the correlation (odds ratio, 1.9; P = 0.00761) of pancreatic cancer risk with PPI exposure. CONCLUSIONS: This investigation revealed in both murine models and human subjects, PPI use is correlated with a risk for development of pancreatic cancer.
Asunto(s)
Neoplasias Pancreáticas , Inhibidores de la Bomba de Protones , Ratones , Humanos , Animales , Inhibidores de la Bomba de Protones/efectos adversos , Ratones Transgénicos , Neoplasias Pancreáticas/inducido químicamente , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Páncreas/metabolismo , Neoplasias PancreáticasRESUMEN
As current interventions for chronic hepatitis B (CHB) rarely induce cure, more effective drugs are needed. Short-term treatment of woodchucks with the novel immunomodulator AIC649, a parapoxvirus-based stimulator of toll-like receptor 9 dependent and independent pathways, has been shown to reduce viral DNA and surface antigen via a unique, biphasic response pattern. The present study evaluated long-term AIC649 treatment in combination with Entecavir for potency and safety in woodchucks. AIC649 monotreatment induced modest reductions in serum viral DNA and surface and e antigens that were associated with the same biphasic response pattern previously observed. Entecavir monotreatment reduced transiently viremia but not antigenemia, while AIC649/Entecavir combination treatment mediated superior viral control. Undetectability of viral antigens and elicitation of antibodies in AIC649/Entecavir-treated woodchucks correlated with the expression of interferons and suppression of viral replication in liver. Combination treatment was well tolerated, and liver enzyme elevations were minor and transient. It was concluded that the AIC649-mediated effects were most likely based on an improvement and/or reconstitution of antiviral immune responses that are typically deficient in CHB. As a combination partner to Entecavir, the antiviral efficacy of AIC649 was markedly enhanced. This preclinical study supports future evaluation of AIC649 for treatment of human CHB.
Asunto(s)
Antivirales/uso terapéutico , Guanina/análogos & derivados , Virus de la Hepatitis B de la Marmota/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Marmota/virología , Animales , ADN Viral/sangre , Modelos Animales de Enfermedad , Drogas en Investigación/uso terapéutico , Guanina/uso terapéutico , Virus de la Hepatitis B de la Marmota/inmunología , Hepatitis B Crónica/inmunología , Resultado del Tratamiento , Viremia/tratamiento farmacológico , Replicación Viral/efectos de los fármacosRESUMEN
Pancreatic cancer is resistant to chemotherapy in part due to the dense desmoplastic fibrosis surrounding the tumor, the immunosuppressive cells in the tumor microenvironment (TME), and the early rate of metastases. In this study, we examined the effects of a CCK receptor antagonist, proglumide, alone and in combination with gemcitabine in murine models of pancreatic cancer. Tumor growth rate, metastases, and survival were assessed in mice bearing syngeneic murine or human pancreatic tumors treated with PBS (control), gemcitabine, proglumide, or the combination of gemcitabine and proglumide. Excised tumors were evaluated histologically for fibrosis, immune cells, molecular markers, and uptake of chemotherapy by mass spectroscopy. Peripheral blood was analyzed with a microRNAs biomarker panel associated with fibrosis and oncogenesis. Differentially expressed genes between tumors of mice treated with gemcitabine monotherapy and combination therapy were compared by RNAseq. When given in combination the two compounds exhibited inhibitory effects by decreasing tumor growth rate by 70%, metastases, and prolonging survival. Proglumide monotherapy altered the TME by decreasing fibrosis, increasing intratumoral CD8+ T-cells, and decreasing arginase-positive cells, thus rendering the tumor sensitive to chemotherapy. Proglumide altered the expression of genes involved in fibrosis, epithelial-mesenchymal transition, and invasion. CCK-receptor antagonism with proglumide renders pancreatic cancer susceptible to chemotherapy.
RESUMEN
Hepatocellular carcinoma (HCC) is the fastest growing cancer worldwide in part due to the obesity epidemic and fatty liver disease, particularly nonalcoholic steatohepatitis (NASH). Chronic inflammation with the release of cytokines and chemokines with activation of hepatic stellate cells results in changes of the liver extracellular matrix (ECM) that predisposes to the development of HCC. Blood levels of the gastrointestinal peptide cholecystokinin (CCK) are increased in humans and mice consuming a high-fat diet. We found that the CCK-B receptor (CCK-BR) expression increased in the livers of mice with NASH. Treatment of mice with a CCK-BR antagonist, proglumide, prevented NASH, lowered hepatic inflammatory cytokines and chemokines, reduced oxidative stress, decreased F4/80+ hepatic macrophages, and prevented HCC. CCK-AR and CCK-BR expression was increased in both murine and human HCC cell lines compared with that of normal liver, and CCK stimulated the growth of wild-type and CCK-A receptor knockout HCC cells in vitro, but not CCK-BR knockout cells suggesting that the CCK-BR mediates proliferation. Proglumide therapy significantly reduced growth by 70% and 73% in mice bearing Dt81Hepa1-6 or in RIL-75 HCC tumors, respectively. IHC of a human liver tissue array with a selective CCK-BR antibody revealed staining of human HCC and no staining in normal liver. PREVENTION RELEVANCE: This investigation demonstrates the role of the gastrointestinal peptide cholecystokinin (CCK) in hepatocellular carcinoma (HCC) and how CCK-BR blockade reverses the premalignant state of the hepatic extracellular matrix hence, rendering it less susceptible to the development of HCC. Thereby, CCK-BR blockade is a novel approach for the prevention/treatment of HCC.
Asunto(s)
Carcinoma Hepatocelular/prevención & control , Neoplasias Hepáticas/prevención & control , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Proglumida/farmacología , Receptor de Colecistoquinina B/antagonistas & inhibidores , Animales , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Quimiocinas CC/genética , Quimiocinas CC/metabolismo , Colecistoquinina/metabolismo , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/patología , Humanos , Hígado/citología , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas/patología , Masculino , Ratones , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/patología , Proglumida/uso terapéutico , Receptor de Colecistoquinina B/genética , Receptor de Colecistoquinina B/metabolismoRESUMEN
OBJECTIVE: The KRAS gene is the most frequently mutated gene in pancreatic cancer, and no successful anti-Ras therapy has been developed. Gastrin has been shown to stimulate pancreatic cancer in an autocrine fashion. We hypothesized that reactivation of the peptide gastrin collaborates with KRAS during pancreatic carcinogenesis. METHODS: LSL-Kras; P48-Cre (KC) mutant KRAS transgenic mice were crossed with gastrin-KO (GKO) mice to develop GKO/KC mice. Pancreata were examined for 8 months for stage of pancreatic intraepithelial neoplasia lesions, inflammation, fibrosis, gastrin peptide, and microRNA expression. Pancreatic intraepithelial neoplasias from mice were collected by laser capture microdissection and subjected to reverse-phase protein microarray, for gastrin and protein kinases associated with signal transduction. Gastrin mRNA was measured by RNAseq in human pancreatic cancer tissues and compared to that in normal pancreas. RESULTS: In the absence of gastrin, PanIN progression, inflammation, and fibrosis were significantly decreased and signal transduction was reversed to the canonical pathway with decreased KRAS. Gastrin re-expression in the PanINs was mediated by miR-27a. Gastrin mRNA expression was significantly increased in human pancreatic cancer samples compared to normal human pancreas controls. CONCLUSIONS: This study supports the mitogenic role of gastrin in activation of KRAS during pancreatic carcinogenesis.
Asunto(s)
Carcinogénesis/genética , Carcinoma in Situ/genética , Gastrinas/genética , Mutación , Páncreas/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Carcinogénesis/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Línea Celular Tumoral , Proliferación Celular/genética , Gastrinas/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Noqueados , Ratones Transgénicos , MicroARNs/genética , Páncreas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) remains the most aggressive malignancy with the lowest 5-year survival rate of all cancers in part owing to the lack of tumor-specific therapy and the rapid metastatic nature of this cancer. The gastrointestinal peptide gastrin is a trophic peptide that stimulates growth of PDAC in an autocrine fashion by interaction with the cholecystokinin receptor that is overexpressed in this malignancy. METHODS: We developed a therapeutic novel polyplex nanoparticle (NP) that selectively targets the cholecystokinin receptor on PDAC. The NP was characterized in vitro and stability testing was performed in human blood. The effects of the target-specific NP loaded with gastrin small interfering RNA (siRNA) was compared with an untargeted NP and with an NP loaded with a scrambled siRNA in vitro and in 2 orthotopic models of PDAC. A polymerase chain reaction metastasis array examined differentially expressed genes from control tumors compared with tumors of mice treated with the targeted polyplex NP. RESULTS: The polyplex NP forms a micelle that safely delivers specific gastrin siRNA to the tumor without off-target toxicity. Consistent with these findings, cellular uptake was confirmed only with the targeted fluorescently labeled NP by confocal microscopy in vitro and by IVIS fluorescent based imaging in mice bearing orthotopic pancreatic cancers but not found with untargeted NPs. Tumor uptake and release of the gastrin siRNA NP was verified by decreased cellular gastrin gene expression by quantitative reverse-transcription polymerase chain reaction and peptide expression by immunohistochemistry. Growth of PDAC was inhibited in a dose-related fashion in cell culture and in vivo. The targeted NP therapy completely blocked tumor metastasis and altered tumor-specific genes. CONCLUSIONS: Our polyplex nanoparticle platform establishes both a strong foundation for the development of receptor-targeted therapeutics and a unique approach for the delivery of siRNA in vivo, thus warranting further exploration of this approach in other types of cancers.
RESUMEN
SB 9200, an orally bioavailable dinucleotide, activates the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) causing the induction of the interferon (IFN) signaling cascade for antiviral defense. The present study evaluated the overall antiviral response in woodchucks upon induction of immune response, first with SB 9200 followed by Entecavir (ETV) versus reduction of viral burden with ETV followed by SB 9200 immunomodulation. Woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated orally with SB 9200 (30 mg/kg/day) and ETV (0.5 mg/kg/day). Group 1 received ETV for 4 weeks followed by SB 9200 for 12 weeks. Group 2 received SB 9200 for 12 weeks followed by ETV for 4 weeks. At the end of treatment in Group 2, average reductions of 6.4 log10 in serum WHV DNA and 3.3 log10 in WHV surface antigen were observed whereas in Group 1, average reductions of 4.2 log10 and 1.1 log10 in viremia and antigenemia were noted. Both groups demonstrated marked reductions in hepatic WHV nucleic acid levels which were more pronounced in Group 2. Following treatment cessation and the 8-week follow-up, recrudescence of viral replication was observed in Group 1 while viral relapse in Group 2 was significantly delayed. The antiviral effects observed in both groups were associated with temporally different induction of IFN-α, IFN-ß, and IFN-stimulated genes in blood and liver. These results suggest that the induction of host immune responses by pretreatment with SB 9200 followed by ETV resulted in antiviral efficacy that was superior to that obtained using the strategy of viral reduction with ETV followed by immunomodulation.
Asunto(s)
Antivirales/uso terapéutico , Guanina/análogos & derivados , Virus de la Hepatitis B de la Marmota/patogenicidad , Marmota/virología , Animales , Guanina/uso terapéutico , Virus de la Hepatitis B de la Marmota/efectos de los fármacos , Virus de la Hepatitis B de la Marmota/inmunología , Hígado/virología , Replicación Viral/efectos de los fármacosRESUMEN
SB 9200, an oral prodrug of the dinucleotide SB 9000, is being developed for the treatment of chronic hepatitis B virus (HBV) infection and represents a novel class of antivirals. SB 9200 is thought to activate the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) resulting in interferon (IFN) mediated antiviral immune responses in virus-infected cells. Additionally, the binding of SB 9200 to these sensor proteins could also sterically block the ability of the viral polymerase to access pre-genomic RNA for nucleic acid synthesis. The immune stimulating and direct antiviral properties of SB 9200 were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) by daily, oral dosing at 15 and 30 mg/kg for 12 weeks. Prolonged treatment resulted in 2.2 and 3.7 log10 reductions in serum WHV DNA and in 0.5 and 1.6 log10 declines in serum WHV surface antigen from pretreatment level with the lower or higher dose of SB 9200, respectively. SB 9200 treatment also resulted in lower hepatic levels of WHV nucleic acids and antigen and reduced liver inflammation. Following treatment cessation, recrudescence of viral replication was observed but with dose-dependent delays in viral relapse. The antiviral effects were associated with dose-dependent and long-lasting induction of IFN-α, IFN-ß and IFN-stimulated genes in blood and liver, which correlated with the prolonged activation of the RIG-I/NOD2 pathway and hepatic presence of elevated RIG-I protein levels. These results suggest that in addition to a direct antiviral activity, SB 9200 induces antiviral immunity during chronic hepadnaviral infection via activation of the viral sensor pathway.