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1.
J Physiol ; 597(12): 3085-3105, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31050811

RESUMEN

KEY POINTS: This work confirms previous reports that CM4620, a small molecule inhibitor of Ca2+ entry via store operated Ca2+ entry (SOCE) channels formed by stromal interaction molecule 1 (STIM1)/Orai complexes, attenuates acinar cell pathology and acute pancreatitis in mouse experimental models. Here we report that intravenous administration of CM4620 reduces the severity of acute pancreatitis in the rat, a hitherto untested species. Using CM4620, we probe further the mechanisms whereby SOCE via STIM1/Orai complexes contributes to the disease in pancreatic acinar cells, supporting a role for endoplasmic reticulum stress/cell death pathways in these cells. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes neutrophil oxidative burst and inflammatory gene expression during acute pancreatitis, including in immune cells which may be either circulating or invading the pancreas. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes activation and fibroinflammatory gene expression within pancreatic stellate cells. ABSTRACT: Key features of acute pancreatitis include excess cellular Ca2+ entry driven by Ca2+ depletion from the endoplasmic reticulum (ER) and subsequent activation of store-operated Ca2+ entry (SOCE) channels in the plasma membrane. In several cell types, including pancreatic acinar, stellate cells (PaSCs) and immune cells, SOCE is mediated via channels composed primarily of Orai1 and stromal interaction molecule 1 (STIM1). CM4620, a selective Orai1 inhibitor, prevents Ca2+ entry in acinar cells. This study investigates the effects of CM4620 in preventing or reducing acute pancreatitis features and severity. We tested the effects of CM4620 on SOCE, trypsinogen activation, acinar cell death, activation of NFAT and NF-κB, and inflammatory responses in ex vivo and in vivo rodent models of acute pancreatitis and human pancreatic acini. We also examined whether CM4620 inhibited cytokine release in immune cells, fibro-inflammatory responses in PaSCs, and oxidative burst in neutrophils, all cell types participating in pancreatitis. CM4620 administration to rats by i.v. infusion starting 30 min after induction of pancreatitis significantly diminished pancreatitis features including pancreatic oedema, acinar cell vacuolization, intrapancreatic trypsin activity, cell death signalling and acinar cell death. CM4620 also decreased myeloperoxidase activity and inflammatory cytokine expression in pancreas and lung tissues, fMLF peptide-induced oxidative burst in human neutrophils, and cytokine production in human peripheral blood mononuclear cells (PBMCs) and rodent PaSCs, indicating that Orai1/STIM1 channels participate in the inflammatory responses of these cell types during acute pancreatitis. These findings support pathological Ca2+ entry-mediated cell death and proinflammatory signalling as central mechanisms in acute pancreatitis pathobiology.


Asunto(s)
Amidinas/uso terapéutico , Antiinflamatorios/uso terapéutico , Bloqueadores de los Canales de Calcio/uso terapéutico , Proteína ORAI1/antagonistas & inhibidores , Pancreatitis/tratamiento farmacológico , Prolina/análogos & derivados , Células Acinares/metabolismo , Amidinas/farmacología , Animales , Antiinflamatorios/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Ceruletida , Citocinas/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Masculino , Ratones Endogámicos C57BL , Células Estrelladas Pancreáticas/metabolismo , Pancreatitis/inducido químicamente , Pancreatitis/inmunología , Pancreatitis/metabolismo , Peroxidasa/metabolismo , Prolina/farmacología , Prolina/uso terapéutico , Ratas , Superóxidos/metabolismo
2.
Gastroenterology ; 153(6): 1674-1686, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-28847752

RESUMEN

BACKGROUND & AIMS: Smoking, an independent risk factor for pancreatitis, accelerates the development of alcoholic pancreatitis. Alcohol feeding of mice induces up-regulation of spliced X-box binding protein 1 (XBP1s), which regulates the endoplasmic reticulum (ER) unfolded protein response and promotes cell survival upon ER stress. We examined whether smoking affects the adaptive mechanisms induced by alcohol and accelerates disorders of the ER in pancreatic acinar cells. METHODS: We studied the combined effects of ethanol (EtOH) and cigarette smoke extract (CSE) on ER stress and cell death responses in mouse and human primary acini and the acinar cell line AR42J. Cells were incubated with EtOH (50 mmol/L), CSE (20-40 µg/mL), or both (CSE+EtOH), and analyzed by immunoblotting, quantitative reverse-transcription polymerase chain reaction, and cell death assays. Some cells were incubated with MKC-3946, an inhibitor of endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1) that blocks XBP1s formation. Male Sprague-Dawley rats were fed isocaloric amounts of an EtOH-containing (Lieber-DeCarli) or control diet for 11 weeks and exposed to cigarette smoke or room air in an exposure chamber for 2 hours each day. During the last 3 weeks, a subset of rats received intravenous injections of lipopolysaccharide (LPS, 3 mg/kg per week) to induce pancreatitis or saline (control). Pancreatic tissues were collected and analyzed by histology and immunostaining techniques. RESULTS: In AR42J and primary acini, CSE+EtOH induced cell death (necrosis and apoptosis), but neither agent alone had this effect. Cell death was associated with a significant decrease in expression of XBP1s. CSE+EtOH, but neither agent alone, slightly decreased adenosine triphosphate levels in AR42J cells, but induced oxidative stress and sustained activation (phosphorylation) of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3, also called PERK) and increased protein levels of DNA damage inducible transcript 3 (DDIT3, also called CHOP). CHOP regulates transcription to promote apoptosis. Incubation of AR42J or primary mouse or human acinar cells with MKC-3946 reduced expression of XBP1s, increased levels of CHOP, and induced cell death. In rats fed an EtOH diet, exposure to cigarette smoke increased ER stress in acinar cells and sensitized the pancreas to LPS-induced pathology. CONCLUSIONS: Cigarette smoke promotes cell death and features of pancreatitis in EtOH-sensitized acinar cells by suppressing the adaptive unfolded protein response signaling pathway. It also activates ER stress pathways that promote acinar cell death.


Asunto(s)
Células Acinares/efectos de los fármacos , Consumo de Bebidas Alcohólicas/efectos adversos , Fumar Cigarrillos/efectos adversos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Etanol/toxicidad , Páncreas Exocrino/efectos de los fármacos , Pancreatitis Alcohólica/etiología , Humo/efectos adversos , Células Acinares/metabolismo , Células Acinares/patología , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Lipopolisacáridos , Masculino , Ratones , Ratones Endogámicos C57BL , Necrosis , Estrés Oxidativo/efectos de los fármacos , Páncreas Exocrino/metabolismo , Páncreas Exocrino/patología , Pancreatitis Alcohólica/metabolismo , Pancreatitis Alcohólica/patología , Ratas Sprague-Dawley , Factores de Riesgo , Factores de Tiempo , Técnicas de Cultivo de Tejidos , Respuesta de Proteína Desplegada/efectos de los fármacos
3.
Am J Pathol ; 187(12): 2726-2743, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28935577

RESUMEN

Knowledge of the molecular mechanisms of acute pancreatitis is largely based on studies using rodents. To assess similar mechanisms in humans, we performed ex vivo pancreatitis studies in human acini isolated from cadaveric pancreata from organ donors. Because data on these human acinar preparations are sparse, we assessed their functional integrity and cellular and organellar morphology using light, fluorescence, and electron microscopy; and their proteome by liquid chromatography-tandem mass spectrometry. Acinar cell responses to the muscarinic agonist carbachol (CCh) and the bile acid taurolithocholic acid 3-sulfate were also analyzed. Proteomic analysis of acini from donors of diverse ethnicity showed similar profiles of digestive enzymes and proteins involved in translation, secretion, and endolysosomal function. Human acini preferentially expressed the muscarinic acetylcholine receptor M3 and maintained physiological responses to CCh for at least 20 hours. As in rodent acini, human acini exposed to toxic concentrations of CCh and taurolithocholic acid 3-sulfate responded with trypsinogen activation, decreased cell viability, organelle damage manifest by mitochondrial depolarization, disordered autophagy, and pathological endoplasmic reticulum stress. Human acini also secreted inflammatory mediators elevated in acute pancreatitis patients, including IL-6, tumor necrosis factor-α, IL-1ß, chemokine (C-C motif) ligands 2 and 3, macrophage inhibitory factor, and chemokines mediating neutrophil and monocyte infiltration. In conclusion, human cadaveric pancreatic acini maintain physiological functions and have similar pathological responses and organellar disorders with pancreatitis-causing treatments as observed in rodent acini.


Asunto(s)
Células Acinares , Técnicas de Cultivo de Célula , Pancreatitis , Células Acinares/citología , Células Acinares/metabolismo , Cadáver , Células Cultivadas , Humanos , Páncreas/citología , Páncreas/metabolismo , Pancreatitis/metabolismo , Pancreatitis/patología , Proteómica
4.
Am J Physiol Gastrointest Liver Physiol ; 311(4): G675-G687, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27609771

RESUMEN

Epidemiological studies support strong links between obesity, diabetes, and pancreatic disorders including pancreatitis and pancreatic adenocarcinoma (PDAC). Type 2 diabetes (T2DM) is associated with insulin resistance, hyperglycemia, and hyperinsulinemia, the latter due to increased insulin secretion by pancreatic beta-cells. We reported that high-fat diet-induced PDAC progression in mice is associated with hyperglycemia, hyperinsulinemia, and activation of pancreatic stellate cells (PaSC). We investigated here the effects of high concentrations of insulin and glucose on mouse and human PaSC growth and fibrosing responses. We found that compared with normal, pancreata from T2DM patients displayed extensive collagen deposition and activated PaSC in islet and peri-islet exocrine pancreas. Mice fed a high-fat diet for up to 12 mo similarly displayed increasing peri-islet fibrosis compared with mice fed control diet. Both quiescent and activated PaSC coexpress insulin (IR; mainly A type) and IGF (IGF-1R) receptors, and both insulin and glucose modulate receptor expression. In cultured PaSC, insulin induced rapid tyrosine autophosphorylation of IR/IGF-1R at specific kinase domain activation loop sites, activated Akt/mTOR/p70S6K signaling, and inactivated FoxO1, a transcription factor that restrains cell growth. Insulin did not promote activation of quiescent PaSC in either 5 mM or 25 mM glucose containing media. However, in activated PaSC, insulin enhanced cell proliferation and augmented production of extracellular matrix proteins, and these effects were abolished by specific inhibition of mTORC1 and mTORC2. In conclusion, our data support the concept that increased local glucose and insulin concentrations associated with obesity and T2DM promote PaSC growth and fibrosing responses.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Diabetes Mellitus Tipo 2/patología , Fibrosis/patología , Glucosa/farmacología , Insulina/farmacología , Células Estrelladas Pancreáticas/efectos de los fármacos , Animales , Células Cultivadas , Colágeno/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa , Femenino , Fibrosis/metabolismo , Humanos , Ratones , Persona de Mediana Edad , Páncreas Exocrino/metabolismo , Páncreas Exocrino/patología , Células Estrelladas Pancreáticas/metabolismo , Células Estrelladas Pancreáticas/patología , Fosforilación/efectos de los fármacos , Receptor IGF Tipo 1/metabolismo
5.
Cancer Cell ; 13(1): 48-57, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18167339

RESUMEN

We investigated whether microRNA expression profiles can predict clinical outcome of NSCLC patients. Using real-time RT-PCR, we obtained microRNA expressions in 112 NSCLC patients, which were divided into the training and testing sets. Using Cox regression and risk-score analysis, we identified a five-microRNA signature for the prediction of treatment outcome of NSCLC in the training set. This microRNA signature was validated by the testing set and an independent cohort. Patients with high-risk scores in their microRNA signatures had poor overall and disease-free survivals compared to the low-risk-score patients. This microRNA signature is an independent predictor of the cancer relapse and survival of NSCLC patients.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , MicroARNs/genética , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Anciano , Carcinoma de Pulmón de Células no Pequeñas/clasificación , Carcinoma de Pulmón de Células no Pequeñas/patología , Estudios de Cohortes , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/clasificación , Neoplasias Pulmonares/patología , Masculino , Invasividad Neoplásica , Estadificación de Neoplasias , Pronóstico , Análisis de Regresión , Reproducibilidad de los Resultados
6.
Mol Ther ; 21(11): 1996-2007, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24081029

RESUMEN

The ERBB receptors are a family of heterodimerization partners capable of driving transformation and metastasis. While the therapeutic targeting of single receptors has proven efficacious, optimal targeting of this receptor family should target all oncogenic members simultaneously. The juxtamembrane domains of ERBB1, ERBB2, and ERBB3 are highly conserved and control various aspects of ERBB-dependent biology. In an effort to block those functions, we have targeted this domain with decoy peptides synthesized in tandem with a cell-penetrating peptide, termed EJ1. Treatment with EJ1 induces cell death, promotes the formation of inactive ERBB multimers, and results in simultaneous reduction of ERBB1, ERBB2, and ERBB3 activation. Treatment also results in the activation of myosin light chain-dependent cell blebbing while inactivating CaMKII signaling, coincident with the induction of cell death. EJ1 also directly translocates to mitochondria, correlating with a loss of mitochondrial membrane potential and production of reactive oxygen species. Finally, treatment of a mouse model of breast cancer with EJ1 results in the inhibition of tumor growth and metastasis without associated toxicities in normal cells. Overall, these data demonstrate that a portion of the ERBB jxm domain, when used as an intracellular decoy, can inhibit tumor growth and metastasis, representing a novel anticancer therapeutic.


Asunto(s)
Antineoplásicos/farmacología , Péptidos de Penetración Celular/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Metástasis de la Neoplasia/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Péptidos de Penetración Celular/uso terapéutico , Progresión de la Enfermedad , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Mamarias Experimentales/secundario , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Multimerización de Proteína/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/química , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Receptor ErbB-3/antagonistas & inhibidores , Receptor ErbB-3/química , Receptor ErbB-3/metabolismo , Transducción de Señal/efectos de los fármacos
7.
Cancers (Basel) ; 16(8)2024 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-38672675

RESUMEN

Diet-induced obesity (DIO) promotes pancreatic ductal adenocarcinoma (PDAC) in mice expressing KRasG12D in the pancreas (KC mice), but the precise mechanisms remain unclear. Here, we performed multiplex quantitative proteomic and phosphoproteomic analysis by liquid chromatography-tandem mass spectrometry and further bioinformatic and spatial analysis of pancreas tissues from control-fed versus DIO KC mice after 3, 6, and 9 months. Normal pancreatic parenchyma and associated proteins were steadily eliminated and the novel proteins, phosphoproteins, and signaling pathways associated with PDAC tumorigenesis increased until 6 months, when most males exhibited cancer, but females did not. Differentially expressed proteins and phosphoproteins induced by DIO revealed the crucial functional role of matrisomal proteins, which implies the roles of upstream regulation by TGFß, extracellular matrix-receptor signaling to downstream PI3K-Akt-mTOR-, MAPK-, and Yap/Taz activation, and crucial effects in the tumor microenvironment such as metabolic alterations and signaling crosstalk between immune cells, cancer-associated fibroblasts (CAFs), and tumor cells. Staining tissues from KC mice localized the expression of several prognostic PDAC biomarkers and elucidated tumorigenic features, such as robust macrophage infiltration, acinar-ductal metaplasia, mucinous PanIN, distinct nonmucinous atypical flat lesions (AFLs) surrounded by smooth muscle actin-positive CAFs, invasive tumors with epithelial-mesenchymal transition arising close to AFLs, and expanding deserted areas by 9 months. We next used Nanostring GeoMX to characterize the early spatial distribution of specific immune cell subtypes in distinct normal, stromal, and PanIN areas. Taken together, these data richly contextualize DIO promotion of Kras-driven PDAC tumorigenesis and provide many novel insights into the signaling pathways and processes involved.

8.
Front Physiol ; 10: 1467, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31849712

RESUMEN

Background: Yes-associated protein 1 (YAP), a transcriptional co-activator and major effector of the Hippo pathway, regulates cell differentiation and morphology in many cell types and supports aberrant tumor growth. Recent studies showed that YAP is expressed in pancreas tissues in pancreatic ductal adenocarcinoma (PDAC) patients and experimental models of PDAC, with YAP largely found in cancer cells and pancreatic stellate cells (PaSC) in the stroma. Methods and Results: We studied here the role of YAP in the activated phenotype of PaSC. We found that YAP is expressed at low levels in normal mouse pancreas, but protein levels significantly increased after pancreas inflammatory damage induced by repeated cerulein administration in wild-type mice or upon initiation of neoplastic transformation of the pancreas parenchyma in Ptf1-Cre;LSL-KrasG12D/+ (KC) mice. In these animal models, YAP upregulation occurred in parallel with activation and proliferation of PaSC. Consistent with these findings, we found robust YAP expression in culture-activated mouse and human PaSC but not in quiescent, freshly isolated cells. Fully activated PaSC isolated from KC mice or PDAC patient tissues exhibited robust nuclear YAP suggesting YAP transcriptional activity. Agents that induce quiescence such as the Bromodomain and Extra-Terminal (BET) inhibitor iBET151 and the p38 MAPK inhibitor SB203580 reduced YAP levels in PaSC. Stimulation of PaSC with the potent mitogen PDGF elicited marked YAP Ser127 phosphorylation. However, unexpectedly, this effect did not diminish YAP nuclear localization, suggesting that YAP phosphorylation at this site does not govern YAP cellular localization in PaSC. siRNA-mediated knockdown of YAP reduced PDGF-induced PaSC expansion in culture and blunted the persistent activation of Akt and ERK elicited by PDGF stimulation, supporting a role for YAP in PDGF-induced cell growth. YAP knockdown also blunted fibroinflammatory gene expression responses both in unstimulated and transforming growth factor beta 1 (TGFß1)-stimulated PaSC. Conclusion: Our data suggest a central role for YAP in sustaining the activated phenotype and fibroinflammatory responses in PaSC. Moreover, our findings indicate that a complex crosstalk between YAP, TGFß1, and PDGF pathways regulates PaSC activity and growth.

9.
Cell Mol Gastroenterol Hepatol ; 5(4): 479-497, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29930975

RESUMEN

BACKGROUND & AIMS: Heavy alcohol drinking is associated with pancreatitis, whereas moderate intake lowers the risk. Mice fed ethanol long term show no pancreas damage unless adaptive/protective responses mediating proteostasis are disrupted. Pancreatic acini synthesize digestive enzymes (largely serine hydrolases) in the endoplasmic reticulum (ER), where perturbations (eg, alcohol consumption) activate adaptive unfolded protein responses orchestrated by spliced X-box binding protein 1 (XBP1). Here, we examined ethanol-induced early structural changes in pancreatic ER proteins. METHODS: Wild-type and Xbp1+/- mice were fed control and ethanol diets, then tissues were homogenized and fractionated. ER proteins were labeled with a cysteine-reactive probe, isotope-coded affinity tag to obtain a novel pancreatic redox ER proteome. Specific labeling of active serine hydrolases in ER with fluorophosphonate desthiobiotin also was characterized proteomically. Protein structural perturbation by redox changes was evaluated further in molecular dynamic simulations. RESULTS: Ethanol feeding and Xbp1 genetic inhibition altered ER redox balance and destabilized key proteins. Proteomic data and molecular dynamic simulations of Carboxyl ester lipase (Cel), a unique serine hydrolase active within ER, showed an uncoupled disulfide bond involving Cel Cys266, Cel dimerization, ER retention, and complex formation in ethanol-fed, XBP1-deficient mice. CONCLUSIONS: Results documented in ethanol-fed mice lacking sufficient spliced XBP1 illustrate consequences of ER stress extended by preventing unfolded protein response from fully restoring pancreatic acinar cell proteostasis during ethanol-induced redox challenge. In this model, orderly protein folding and transport to the secretory pathway were disrupted, and abundant molecules including Cel with perturbed structures were retained in ER, promoting ER stress-related pancreas pathology.

10.
Gastroenterol Res Pract ; 2017: 9505460, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29118810

RESUMEN

BACKGROUND: Environmental factors and genetic mutations have been increasingly recognized as risk factors for chronic pancreatitis (CP). The PRSS1 p.R122H mutation was the first discovered to affect hereditary CP, with 80% penetrance. We performed here a systematic review and meta-analysis to evaluate the associations of PRSS1 p.R122H mutation with CP of diverse etiology. METHODS: The PubMed, EMBASE, and MEDLINE database were reviewed. The pooled odds ratio (OR) with 95% confidence intervals was used to evaluate the association of p.R122H mutation with CP. Initial analysis was conducted with all etiologies of CP, followed by a subgroup analysis for hereditary and nonhereditary CP, including alcoholic or idiopathic CP. RESULTS: A total of eight case-control studies (1733 cases and 2415 controls) were identified and included. Overall, PRSS1 p.R122H mutation was significantly associated with an increased risk of CP (OR = 4.78[1.13-20.20]). Further analysis showed p.R122H mutation strongly associated with the increased risk of hereditary CP (OR = 65.52[9.09-472.48]) but not with nonhereditary CP, both alcoholic and idiopathic CP. CONCLUSIONS: Our study showing the differential role of p.R122H mutation in various etiologies of CP indicates that this complex disorder is likely influenced by multiple genetic factors as well as environmental factors.

11.
PLoS One ; 12(9): e0184455, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28886117

RESUMEN

Epidemiologic data has linked obesity to a higher risk of pancreatic cancer, but the underlying mechanisms are poorly understood. To allow for detailed mechanistic studies in a relevant model mimicking diet-induced obesity and pancreatic cancer, a high-fat, high-calorie diet (HFCD) was given to P48+/Cre;LSL-KRASG12D (KC) mice carrying a pancreas-specific oncogenic Kras mutation. The mice were randomly allocated to a HFCD or control diet (CD). Cohorts were sacrificed at 3, 6, and 9 months and tissues were harvested for further analysis. Compared to CD-fed mice, HFCD-fed animals gained significantly more weight. Importantly, the cancer incidence was remarkably increased in HFCD-fed KC mice, particularly in male KC mice. In addition, KC mice fed the HFCD showed more extensive inflammation and fibrosis, and more advanced PanIN lesions in the pancreas, compared to age-matched CD-fed animals. Interestingly, we found that the HFCD reduced autophagic flux in PanIN lesions in KC mice. Further, exome sequencing of isolated murine PanIN lesions identified numerous genetic variants unique to the HFCD. These data underscore the role of sustained inflammation and dysregulated autophagy in diet-induced pancreatic cancer development and suggest that diet-induced genetic alterations may contribute to this process. Our findings provide a better understanding of the mechanisms underlying the obesity-cancer link in males and females, and will facilitate the development of interventions targeting obesity-associated pancreatic cancer.


Asunto(s)
Dieta Alta en Grasa/efectos adversos , Ingestión de Energía , Mutación , Neoplasias Pancreáticas/etiología , Proteínas Proto-Oncogénicas p21(ras)/genética , Sustitución de Aminoácidos , Animales , Autofagia/genética , Peso Corporal , Codón , Biología Computacional/métodos , Modelos Animales de Enfermedad , Exoma , Matriz Extracelular/metabolismo , Femenino , Fibrosis , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Inflamación/etiología , Inflamación/patología , Masculino , Ratones , Neoplasias Pancreáticas/patología
12.
PLoS One ; 11(2): e0148999, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26849807

RESUMEN

Activated pancreatic stellate cells (PaSC) are key participants in the stroma of pancreatic cancer, secreting extracellular matrix proteins and inflammatory mediators. Tumors are poorly vascularized, creating metabolic stress conditions in cancer and stromal cells that necessitate adaptive homeostatic cellular programs. Activation of autophagy and the endoplasmic reticulum unfolded protein response (UPR) have been described in hepatic stellate cells, but the role of these processes in PaSC responses to metabolic stress is unknown. We reported that the PI3K/mTOR pathway, which AMPK can regulate through multiple inputs, modulates PaSC activation and fibrogenic potential. Here, using primary and immortalized mouse PaSC, we assess the relative contributions of AMPK/mTOR signaling, autophagy and the UPR to cell fate responses during metabolic stress induced by mitochondrial dysfunction. The mitochondrial uncoupler rottlerin at low doses (0.5-2.5 µM) was added to cells cultured in 10% FBS complete media. Mitochondria rapidly depolarized, followed by altered mitochondrial dynamics and decreased cellular ATP levels. This mitochondrial dysfunction elicited rapid, sustained AMPK activation, mTOR pathway inhibition, and blockade of autophagic flux. Rottlerin treatment also induced rapid, sustained PERK/CHOP UPR signaling. Subsequently, high doses (>5 µM) induced loss of cell viability and cell death. Interestingly, AMPK knock-down using siRNA did not prevent rottlerin-induced mTOR inhibition, autophagy, or CHOP upregulation, suggesting that AMPK is dispensable for these responses. Moreover, CHOP genetic deletion, but not AMPK knock-down, prevented rottlerin-induced apoptosis and supported cell survival, suggesting that UPR signaling is a major modulator of cell fate in PaSC during metabolic stress. Further, short-term rottlerin treatment reduced both PaSC fibrogenic potential and IL-6 mRNA expression. In contrast, expression levels of the angiogenic factors HGF and VEGFα were unaffected, and the immune modulator IL-4 was markedly upregulated. These data imply that metabolic stress-induced PaSC reprogramming differentially modulates neighboring cells in the tumor microenvironment.


Asunto(s)
Autofagia , Mitocondrias/metabolismo , Páncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Transducción de Señal , Microambiente Tumoral , Respuesta de Proteína Desplegada , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Activación Enzimática/genética , Interleucina-4/biosíntesis , Interleucina-4/genética , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/patología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Páncreas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Regulación hacia Arriba/genética , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismo
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