RESUMEN
Nonalcoholic fatty liver disease (NAFLD) is an increasingly common condition, affecting up to 25% of the population worldwide. NAFLD has been linked to several conditions, including hepatic inflammation, fibrosis, and hepatocellular carcinoma (HCC), however the role of NAFLD in cholangitis and the development of cholangiocellular carcinoma (CCC) remains poorly understood. This study investigated whether a high-fat diet (HFD) promotes cholangitis and the development of CCC in mice. We used liver-specific E-cadherin gene (CDH1) knockout mice, CDH1∆Liv , which develop spontaneous inflammation in the portal areas along with periductal onion skin-like fibrosis, similar to that of primary sclerosing cholangitis (PSC). An HFD or normal diet (ND) was fed to CDH1∆Liv mice for 7 mo. In addition, CDH1∆Liv mice were crossed with LSL-KrasG12D mice, fed an HFD, and assessed in terms of liver tumor development. The extent of cholangitis and number of bile ductules significantly increased in mice fed an HFD compared with ND-administered CDH1∆Liv mice. The numbers of Sox9 and CD44-positive stem cell-like cells were significantly increased in HFD mice. LSL-KrasG12D /CDH1∆Liv HFD mice exhibited increased aggressiveness along with the development of numerous HCC and CCC, whereas LSL-KrasG12D /CDH1∆Liv ND mice showed several macroscopic tumors with both HCC and CCC components. In conclusion, NAFLD exacerbates cholangitis and promotes the development of both HCC and CCC in mice.
Asunto(s)
Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/patología , Colangitis/patología , Enfermedad del Hígado Graso no Alcohólico/patología , Animales , Antígenos CD/metabolismo , Neoplasias de los Conductos Biliares/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Colangiocarcinoma/metabolismo , Colangitis/metabolismo , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Inflamación/metabolismo , Inflamación/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
BACKGROUND & AIMS: The intestinal epithelium is maintained by long-lived intestinal stem cells (ISCs) that reside near the crypt base. Above the ISC zone, there are short-lived progenitors that normally give rise to lineage-specific differentiated cell types but can dedifferentiate into ISCs in certain circumstances. However, the role of epithelial dedifferentiation in cancer development has not been fully elucidated. METHODS: We performed studies with Bhlha15-CreERT, Lgr5-DTR-GFP, Apcflox/flox, LSL-Notch (IC), and R26-reporter strains of mice. Some mice were given diphtheria toxin to ablate Lgr5-positive cells, were irradiated, or were given 5-fluorouracil, hydroxyurea, doxorubicin, or dextran sodium sulfate to induce intestinal or colonic tissue injury. In intestinal tissues, we analyzed the fate of progeny that expressed Bhlha15. We used microarrays and reverse-transcription PCR to analyze gene expression patterns in healthy and injured intestinal tissues and in tumors. We analyzed gene expression patterns in human colorectal tumors using The Cancer Genome Atlas data set. RESULTS: Bhlha15 identified Paneth cells and short-lived secretory precursors (including pre-Paneth label-retaining cells) located just above the ISC zone in the intestinal epithelium. Bhlha15+ cells had no plasticity after loss of Lgr5-positive cells or irradiation. However, Bhlha15+ secretory precursors started to supply the enterocyte lineage after doxorubicin-induced epithelial injury in a Notch-dependent manner. Sustained activation of Notch converts Bhlha15+ secretory precursors to long-lived enterocyte progenitors. Administration of doxorubicin and expression of an activated form of Notch resulted in a gene expression pattern associated with enterocyte progenitors, whereas only sustained activation of Notch altered gene expression patterns in Bhlha15+ precursors toward those of ISCs. Bhlha15+ enterocyte progenitors with sustained activation of Notch formed intestinal tumors with serrated features in mice with disruption of Apc. In the colon, Bhlha15 marked secretory precursors that became stem-like, cancer-initiating cells after dextran sodium sulfate-induced injury, via activation of Src and YAP signaling. In analyses of human colorectal tumors, we associated activation of Notch with chromosome instability-type tumors with serrated features in the left colon. CONCLUSIONS: In mice, we found that short-lived precursors can undergo permanent reprogramming by activation of Notch and YAP signaling. These cells could mediate tumor formation in addition to traditional ISCs.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias del Colon/genética , Enterocitos/patología , Mucosa Intestinal/metabolismo , Receptores Notch/metabolismo , Células Madre/metabolismo , Transcriptoma , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Hormonales/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Antígeno CD24/metabolismo , Proteínas de Unión al Calcio , Proteínas de Ciclo Celular , Plasticidad de la Célula , Cromogranina A/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Doxorrubicina/farmacología , Enterocitos/metabolismo , Expresión Génica , Perfilación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Intestino Delgado/citología , Intestino Delgado/metabolismo , Ratones , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Proteínas Asociadas a Pancreatitis , Células de Paneth , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Células Madre/efectos de los fármacos , Células Madre/fisiología , Células Madre/efectos de la radiación , Tamoxifeno/farmacología , Proteínas Señalizadoras YAP , Familia-src Quinasas/metabolismoRESUMEN
Chronic inflammation and intestinal metaplasia are strongly associated with gastric carcinogenesis. Kras activation and Pten deletion are observed in intestinal-type gastric cancer, and Cdh1 mutation is associated with diffuse-type gastric cancer. Although various mouse models of gastric carcinogenesis have been reported, few mouse lines enable gene manipulation selectively in the stomach. Here we established a Tff1-Cre bacterial artificial chromosome transgenic mouse line in an attempt to induce gene modification specifically in the gastric pit lineage. In the stomach, Tff1-Cre-mediated recombination was most evident in the pit lineage in the corpus and in entire antral glands; recombination was also observed in a few gastric chief and parietal cells. Outside the stomach, recombination was patchy throughout the intestines, and particularly frequently in the duodenum (Brunner glands), cecum, and proximal colon. In the stomachs of Tff1-Cre;LSL-KrasG12D mice, proliferating cell clusters expanded throughout the corpus glands, with foveolar cell expansion with ectopic Alcian blue-positive mucins, oxyntic atrophy, and pseudopyloric changes with spasmolytic polypeptide-expressing metaplasia; however, gastric cancer was not observed even at 12 months of age. Corpus-derived organoids from Tff1-Cre;LSL-KrasG12D mice exhibited accelerated growth and abnormal differentiation with a loss of chief and parietal cell markers. Tff1-Cre;Ptenflox/flox mice displayed similar changes to those seen in Tff1-Cre;LSL-KrasG12D mice, both with aberrant ERK activation within 3 months. In contrast, Tff1-Cre;Cdh1flox/flox mice initially showed signet ring-like cells that were rapidly lost with disruption of the mucosal surface, and later developed gastric epithelial shedding with hyperproliferation and loss of normal gastric lineages. Eventually, the glandular epithelium in Tff1-Cre;Cdh1flox/flox mice was completely replaced by squamous epithelium which expanded from the forestomach. Tff1-Cre mice offer an additional useful tool for studying gastric carcinogenesis both in vivo and in vitro. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Cadherinas/deficiencia , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Mucosa Gástrica/enzimología , Gastritis/enzimología , Fosfohidrolasa PTEN/deficiencia , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias Gástricas/enzimología , Animales , Cadherinas/genética , Linaje de la Célula , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Cromosomas Artificiales Bacterianos , Mucinas Gástricas/genética , Mucinas Gástricas/metabolismo , Mucosa Gástrica/patología , Gastritis/genética , Gastritis/patología , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Integrasas/genética , Metaplasia , Ratones Transgénicos , Fosfohidrolasa PTEN/genética , Fenotipo , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Técnicas de Cultivo de Tejidos , Factor Trefoil-1/genéticaRESUMEN
The carcinogenic mechanism of extrahepatic cholangiocarcinoma (ECC) is unclear, due at least in part to the lack of an appropriate mouse model. Because human studies have reported frequent genetic alterations in the Ras- and TGFß/SMAD-signaling pathways in ECC, mice with tamoxifen-inducible, duct-cell-specific Kras activation and a TGFß receptor type 2 (TGFßR2) deletion were first generated by crossing LSL-KrasG12D , Tgfbr2flox/flox , and K19CreERT mice (KT-K19CreERT ). However, KT-K19CreERT mice showed only mild hyperplasia of biliary epithelial cells (BECs) in the extrahepatic bile duct (EHBD) and died within 7 wk, probably a result of lung adenocarcinomas. Next, to analyze the additional effect of E-cadherin loss, KT-K19CreERT mice were crossed with CDH1flox/flox mice (KTC-K19CreERT ). Surprisingly, KTC-K19CreERT mice exhibited a markedly thickened EHBD wall accompanied by a swollen gallbladder within 4 wk after tamoxifen administration. Histologically, invasive periductal infiltrating-type ECC with lymphatic metastasis was observed. Time-course analysis of EHBD revealed that recombined BECs lining the bile duct lumen detached due to E-cadherin loss, whereas recombined cells could survive in the peribiliary glands (PBGs), which are considered a BEC stem-cell niche. Detached dying BECs released high levels of IL-33, as determined by microarray analysis using biliary organoids, and stimulated inflammation and a regenerative response by PBGs, leading eventually to ECC development. Cell lineage tracing suggested PBGs as the cellular origin of ECC. IL-33 cooperated with Kras and TGFßR2 mutations in the development of ECC, and anti-IL-33 treatment suppressed ECC development significantly. Thus, this mouse model provided insight into the carcinogenic mechanisms, cellular origin, and potential therapeutic targets of ECC.
Asunto(s)
Neoplasias de los Conductos Biliares/metabolismo , Conductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Interleucina-33/metabolismo , Animales , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Conductos Biliares/lesiones , Conductos Biliares/patología , Cadherinas/genética , Cadherinas/metabolismo , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Epitelio/lesiones , Epitelio/metabolismo , Epitelio/patología , Interleucina-33/genética , Ratones , Ratones Transgénicos , Mutación , Metástasis de la Neoplasia , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismoRESUMEN
OBJECTIVE: Metabolic reprogramming of tumour cells that allows for adaptation to their local environment is a hallmark of cancer. Interestingly, obesity-driven and non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) mouse models commonly exhibit strong steatosis in tumour cells as seen in human steatohepatitic HCC (SH-HCC), which may reflect a characteristic metabolic alteration. DESIGN: Non-tumour and HCC tissues obtained from diethylnitrosamine-injected mice fed either a normal or a high-fat diet (HFD) were subjected to comprehensive metabolome analysis, and the significance of obesity-mediated metabolic alteration in hepatocarcinogenesis was evaluated. RESULTS: The extensive accumulation of acylcarnitine species was seen in HCC tissues and in the serum of HFD-fed mice. A similar increase was found in the serum of patients with NASH-HCC. The accumulation of acylcarnitine could be attributed to the downregulation of carnitine palmitoyltransferase 2 (CPT2), which was also seen in human SH-HCC. CPT2 downregulation induced the suppression of fatty acid ß-oxidation, which would account for the steatotic changes in HCC. CPT2 knockdown in HCC cells resulted in their resistance to lipotoxicity by inhibiting the Src-mediated JNK activation. Additionally, oleoylcarnitine enhanced sphere formation by HCC cells via STAT3 activation, suggesting that acylcarnitine accumulation was a surrogate marker of CPT2 downregulation and directly contributed to hepatocarcinogenesis. HFD feeding and carnitine supplementation synergistically enhanced HCC development accompanied by acylcarnitine accumulation in vivo. CONCLUSION: In obesity-driven and NASH-driven HCC, metabolic reprogramming mediated by the downregulation of CPT2 enables HCC cells to escape lipotoxicity and promotes hepatocarcinogenesis.
Asunto(s)
Carcinoma Hepatocelular/etiología , Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina/análogos & derivados , Neoplasias Hepáticas/etiología , Enfermedad del Hígado Graso no Alcohólico/sangre , Obesidad/complicaciones , Adulto , Anciano , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carnitina/metabolismo , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Femenino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/metabolismo , Obesidad/patologíaRESUMEN
During human gastric carcinogenesis, intestinal metaplasia is frequently seen in the atrophic stomach. In mice, a distinct type of metaplasia known as spasmolytic polypeptide-expressing metaplasia (SPEM) is found in several inflammatory and genetically engineered models. Given the diversity of long- and short-term models of mouse SPEM, it remains unclear whether all models have a shared or distinct molecular mechanism. The origin of SPEM in mice is presently under debate. It is postulated that stem or progenitor cells acquire genetic alterations that then supply metaplastic cell clones, whereas the possibility of transdifferentiation or dedifferentiation from mature gastric chief cells has also been suggested. In this study, we report that loss of chief cells was sufficient to induce short-term regenerative SPEM-like lesions that originated from chief cell precursors in the gastric neck region. Furthermore, Lgr5+ mature chief cells failed to contribute to both short- and long-term metaplasia, whereas isthmus stem and progenitor cells efficiently contributed to long-term metaplasia. Interestingly, multiple administrations of high-dose pulsed tamoxifen induced expansion of Lgr5 expression and Lgr5-CreERT recombination within the isthmus progenitors apart from basal chief cells. Thus we conclude that short-term SPEM represents a regenerative process arising from neck progenitors following chief cell loss, whereas true long-term SPEM originates from isthmus progenitors. Mature gastric chief cells may be dispensable for SPEM development. NEW & NOTEWORTHY Recently, dedifferentiation ability in gastric chief cells during metaplasia development has been proposed. Our findings reveal that lesions that were thought to be acute metaplasia in fact represent normal regeneration supplied from neck lineage and that isthmus stem/progenitors are more responsible for sustained metaplastic changes. Cellular plasticity in gastric chief cells may be more limited than recently highlighted.
Asunto(s)
Carcinogénesis , Células Principales Gástricas , Péptidos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Gástricas , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Linaje de la Célula , Transdiferenciación Celular/fisiología , Células Principales Gástricas/metabolismo , Células Principales Gástricas/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Metaplasia , Ratones , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/patología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a life-threatening disease and there is an urgent need to develop improved therapeutic approaches. The role of c-Jun N-terminal kinase (JNK) in PDAC stroma is not well defined even though dense desmoplastic reactions are characteristic of PDAC histology. We aimed to explore the role of JNK in PDAC stroma in mice. We crossed Ptf1aCre/+ ;KrasG12D/+ mice with JNK1-/- mice to generate Ptf1aCre/+ ;KrasG12D/+ ;JNK1-/- (Kras;JNK1-/- ) mice. Tumor weight was significantly lower in Kras;JNK1-/- mice than in Kras;JNK1+/- mice, whereas histopathological features were similar. We also transplanted a murine PDAC cell line (mPC) with intact JNK1 s.c. into WT and JNK1-/- mice. Tumor diameters were significantly smaller in JNK1-/- mice. Phosphorylated JNK (p-JNK) was activated in α-smooth muscle actin (SMA)-positive cells in tumor stroma, and mPC-conditioned medium activated p-JNK in tumor-associated fibroblasts (TAF) in vitro. Relative expression of Ccl20 was downregulated in stimulated TAF. Ccl20 is an important chemokine that promotes CD8+ T-cell infiltration by recruitment of dendritic cells, and the number of CD8+ T cells was decreased in Kras;JNK1+/- mice compared with Kras;JNK1-/- mice. These results suggest that the cancer secretome decreases Ccl20 secretion from TAF by activation of JNK, and downregulation of Ccl20 secretion might be correlated with reduction of infiltrating CD8+ T cells. Therefore, we concluded that inhibition of activated JNK in pancreatic tumor stroma could be a potential therapeutic target to increase Ccl20 secretion from TAF and induce accumulation of CD8+ T cells, which would be expected to enhance antitumor immunity.
Asunto(s)
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Quimiocina CCL20/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de Transcripción/genética , Adenocarcinoma/patología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma Ductal Pancreático/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Mutación , Células Madre Neoplásicas/patología , Transducción de Señal/genéticaRESUMEN
E-cadherin is an important adhesion molecule whose loss is associated with progression and poor prognosis of liver cancer. However, it is unclear whether the loss of E-cadherin is a real culprit or a bystander in liver cancer progression. In addition, the precise role of E-cadherin in maintaining liver homeostasis is also still unknown, especially in vivo. Here we demonstrate that liver-specific E-cadherin knockout mice develop spontaneous periportal inflammation via an impaired intrahepatic biliary network, as well as periductal fibrosis, which resembles primary sclerosing cholangitis. Inducible gene knockout studies identified E-cadherin loss in biliary epithelial cells as a causal factor of cholangitis induction. Furthermore, a few of the E-cadherin knockout mice developed spontaneous liver cancer. When knockout of E-cadherin is combined with Ras activation or chemical carcinogen administration, E-cadherin knockout mice display markedly accelerated carcinogenesis and an invasive phenotype associated with epithelial-mesenchymal transition, up-regulation of stem cell markers, and elevated ERK activation. Also in human hepatocellular carcinoma, E-cadherin loss correlates with increased expression of mesenchymal and stem cell markers, and silencing of E-cadherin in hepatocellular carcinoma cell lines causes epithelial-mesenchymal transition and increased invasiveness, suggesting that E-cadherin loss can be a causal factor of these phenotypes. Thus, E-cadherin plays critical roles in maintaining homeostasis and suppressing carcinogenesis in the liver.
Asunto(s)
Cadherinas/metabolismo , Carcinogénesis , Colangitis Esclerosante/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Colangitis/metabolismo , Transición Epitelial-Mesenquimal , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hepatocitos/citología , Inflamación , Hígado/patología , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Metástasis de la Neoplasia , Fenotipo , Pronóstico , Células Madre/citologíaRESUMEN
Histopathological changes of the gastric mucosa after Helicobacter pylori infection, such as atrophy, metaplasia, and dysplasia, are considered to be precursors of gastric cancer, yet the mechanisms of histological progression are unknown. The aim of this study was to analyze the histopathological features of the gastric mucosa in mice infected with H. pylori strain PMSS1 in relation to gastric stem cell marker expression. C57BL/6J mice infected with PMSS1 were examined for histopathological changes, levels of proinflammatory cytokines, and expression of stem cell markers. Histopathological gastritis scores, such as atrophy and metaplasia, and levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß), were increased after PMSS1 infection. Expression levels of the cell proliferation and stem cell markers CD44 and SOX9 were also significantly increased in PMSS1-infected mice. Importantly, almost all metaplastic cells induced by PMSS1 infection expressed SOX9. When IL-1 receptor (IL-1R) knockout mice were infected with PMSS1, metaplastic changes and expression levels of stem cell markers were significantly decreased compared with those in wild-type (WT) mice. In conclusion, H. pylori infection induced the expression of cytokines and stem cell markers and histopathological metaplasia in the mouse gastric mucosa. SOX9 expression, in particular, was strongly associated with metaplastic changes, and these changes were dependent on IL-1 signaling. The results suggested the importance of SOX9 in gastric carcinogenesis.
Asunto(s)
Mucosa Gástrica/patología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/patología , Helicobacter pylori/patogenicidad , Interleucina-1/metabolismo , Factor de Transcripción SOX9/genética , Animales , Citocinas/genética , Citocinas/inmunología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/ultraestructura , Gastritis/inmunología , Gastritis/metabolismo , Gastritis/microbiología , Gastritis/patología , Infecciones por Helicobacter/inmunología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/inmunología , Receptores de Hialuranos/genética , Interleucina-1/deficiencia , Interleucina-1/genética , Interleucina-1/inmunología , Metaplasia/microbiología , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
BACKGROUND: Although some molecularly targeted drugs for colorectal cancer are used clinically and contribute to a better prognosis, the current median survival of advanced colorectal cancer patients is not sufficient. Autophagy, a basic cell survival mechanism mediated by recycling of cellular amino acids, plays an important role in cancer. Recently, autophagy has been highlighted as a promising new molecular target. The unfolded protein response (UPR) reportedly act in complementary fashion with autophagy in intestinal homeostasis. However, the roles of UPR in colon cancer under autophagic inhibition remain to be elucidated. We aim to clarify the inhibitory effect of autophagy on colon cancer. METHODS: We crossed K19 (CreERT) and Atg5 (flox/flox) mice to generate Atg5 (flox/flox)/K19 (CreERT) mice. Atg5 (flox/flox)/K19 (CreERT) mice were first treated with azoxymethane/dextran sodium sulfate and then injected with tamoxifen to inhibit autophagy in CK19-positive epithelial cells. To examine the anti-cancer mechanisms of autophagic inhibition, we used colon cancer cell lines harboring different p53 gene statuses, as well as small interfering RNAs (siRNAs) targeting Atg5 and immunoglobulin heavy-chain binding protein (BiP), a chaperone to aid folding of unfolded proteins. RESULTS: Colon tumors in Atg5 (flox/flox)/K19 (CreERT) mice showed loss of autophagic activity and decreased tumor size (the total tumor diameter was 28.1 mm in the control and 20.7 mm in Atg5 (flox/flox)/K19 (CreERT) mice, p = 0.036). We found that p53 and UPR/endoplasmic reticulum (ER) stress-related proteins, such as cleaved caspase 3, and CAAT/enhancer-binding protein homologous protein, are up-regulated in colon tumors of Atg5 (flox/flox)/K19 (CreERT) mice. Although Atg5 and BiP silencing, respectively, increased apoptosis in p53 wild type cells, Atg5 silencing alone did not show the same effect on apoptosis in p53 mutant cells. However, co-transfection of Atg5 and BiP siRNAs led to increased apoptosis in p53 mutant cells. CONCLUSIONS: Blocking autophagy has potential in the treatment of colon cancer by inducing apoptosis via p53 and ER stress, and suppressing the UPR pathway is a valid strategy to overcome resistance to autophagic inhibition.
Asunto(s)
Apoptosis/fisiología , Autofagia/fisiología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/prevención & control , Estrés del Retículo Endoplásmico/fisiología , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Línea Celular Tumoral , Genes p53/fisiología , Células HCT116 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
UNLABELLED: MicroRNAs (miRNAs) are small RNAs that regulate the expression of specific target genes. While deregulated miRNA expression levels have been detected in many tumors, whether miRNA functional impairment is also involved in carcinogenesis remains unknown. We investigated whether deregulation of miRNA machinery components and subsequent functional impairment of miRNAs are involved in hepatocarcinogenesis. Among miRNA-containing ribonucleoprotein complex components, reduced expression of DDX20 was frequently observed in human hepatocellular carcinomas, in which enhanced nuclear factor-κB (NF-κB) activity is believed to be closely linked to carcinogenesis. Because DDX20 normally suppresses NF-κB activity by preferentially regulating the function of the NF-κB-suppressing miRNA-140, we hypothesized that impairment of miRNA-140 function may be involved in hepatocarcinogenesis. DNA methyltransferase 1 (Dnmt1) was identified as a direct target of miRNA-140, and increased Dnmt1 expression in DDX20-deficient cells hypermethylated the promoters of metallothionein genes, resulting in decreased metallothionein expression leading to enhanced NF-κB activity. MiRNA-140-knockout mice were prone to hepatocarcinogenesis and had a phenotype similar to that of DDX20 deficiency, suggesting that miRNA-140 plays a central role in DDX20 deficiency-related pathogenesis. CONCLUSION: These results indicate that miRNA-140 acts as a liver tumor suppressor, and that impairment of miRNA-140 function due to a deficiency of DDX20, a miRNA machinery component, could lead to hepatocarcinogenesis.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Proteína 20 DEAD-Box/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , FN-kappa B/metabolismo , Animales , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1 , Células Hep G2 , Humanos , Metalotioneína/metabolismo , Ratones , Ratones Noqueados , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular functions and are highly active in many types of human cancers. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream MAPK involved in apoptosis, inflammation, and carcinogenesis. This study investigated the role of ASK1 in the development of gastric cancer. In human gastric cancer specimens, we observed increased ASK1 expression, compared to nontumor epithelium. Using a chemically induced murine gastric tumorigenesis model, we observed increased tumor ASK1 expression, and ASK1 knockout mice had both fewer and smaller tumors than wild-type (WT) mice. ASK1 siRNA inhibited cell proliferation through the accumulation of cells in G1 phase of the cell cycle, and reduced cyclin D1 expression in gastric cancer cells, whereas these effects were uncommon in other cancer cells. ASK1 overexpression induced the transcription of cyclin D1, through AP-1 activation, and ASK1 levels were regulated by cyclin D1, via the Rb-E2F pathway. Exogenous ASK1 induced cyclin D1 expression, followed by elevated expression of endogenous ASK1. These results indicate an autoregulatory mechanism of ASK1 in the development of gastric cancer. Targeting this positive feedback loop, ASK1 may present a potential therapeutic target for the treatment of advanced gastric cancer.
Asunto(s)
Apoptosis , Ciclina D1/metabolismo , Regulación Neoplásica de la Expresión Génica , MAP Quinasa Quinasa Quinasa 5/metabolismo , Neoplasias Gástricas/metabolismo , Animales , Ciclo Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Factores de Transcripción E2F/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de SeñalRESUMEN
c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family, and it is reportedly involved in the development of several cancers. However, the role of JNK in pancreatic cancer has not been elucidated. We assessed t he involvement of JNK in the development of pancreatic cancer and investigated the therapeutic effect of JNK inhibitors on this deadly cancer. Small interfering RNAs against JNK or the JNK inhibitor SP600125 were used to examine the role of JNK in cellular proliferation and the cell cycles of pancreatic cancer cell lines. Ptf1a(cre/+) ;LSL-Kras(G12D/+) ;Tgfbr2(flox/flox) mice were treated with the JNK inhibitor to examine pancreatic histology and survival. The effect of JNK inhibition on tumor angiogenesis was also assessed using cell lines and murine pancreatic cancer specimens. JNK was frequently activated in human and murine pancreatic cancer in vitro and in vivo. Growth of human pancreatic cancer cell lines was suppressed by JNK inhibition through G1 arrest in the cell cycle with decreased cyclin D1 expression. In addition, oncogenic K-ras expression led to activation of JNK in pancreatic cancer cell lines. Treatment of Ptf1a(cre/+) ;LSL-Kras(G12D/+) ;Tgfbr2(flox/flox) mice with the JNK inhibitor decreased growth of murine pancreatic cancer and prolonged survival of the mice significantly. Angiogenesis was also decreased by JNK inhibition in vitro and in vivo. In conclusion, activation of JNK promotes development of pancreatic cancer, and JNK may be a potential therapeutic target for pancreatic cancer.
Asunto(s)
Adenocarcinoma/metabolismo , Antracenos/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/genética , Animales , Línea Celular Tumoral , Proliferación Celular , Silenciador del Gen , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidadRESUMEN
UNLABELLED: The stress-activated mitogen-activated protein kinases (MAPKs), c-Jun NH2-terminal kinase (JNK), and p38 have been implicated in hepatocarcinogenesis. Although the many interrelated functions of JNK and p38 are precisely regulated by upstream signaling molecules, little is known about upstream regulators. We investigated the role of apoptosis signal-regulating kinase 1 (ASK1), a major player in the regulation of JNK and p38 activities, in hepatocarcinogenesis using a mouse hepatocellular carcinoma (HCC) model. ASK1-deficient (ASK1(-/-) ) and wildtype (WT) mice were treated with diethylnitrosamine on postnatal day 14. Strikingly, after 7 months, approximately three times as many tumors developed in ASK1(-/-) mice as in WT mice. Although JNK and p38 activation were attenuated in ASK1(-/-) HCCs relative to WT HCCs, cell proliferation was comparable in HCCs from both types of mice. On the other hand, both cancer cell apoptosis and hyperphosphorylation of BimEL, a proapoptotic Bcl-2 family member, were suppressed in the ASK1(-/-) HCCs. ASK1(-/-) mice showed remarkable resistance to Fas-induced hepatocyte apoptosis in vivo, probably because of attenuated JNK-mediated BimEL phosphorylation and mitochondrial apoptotic pathway activation. The reintroduction of ASK1 to ASK1(-/-) mouse liver using an adenoviral vector restored Fas-induced hepatocyte death and phosphorylation of JNK and BimEL. Similar findings were obtained in tumor necrosis factor alpha-induced hepatocyte apoptosis. Furthermore, ASK1 was involved in DNA damage-induced p21 up-regulation through a p38 pathway. CONCLUSION: ASK1 is involved in death receptor-mediated apoptosis and DNA-damage response by way of stress-activated MAPK in the liver, and thus acts as a tumor suppressor in hepatocarcinogenesis. This study provides new insight into the regulation of stress- activated MAPK signaling in hepatocarcinogenesis.
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Carcinoma Hepatocelular/prevención & control , Neoplasias Hepáticas/prevención & control , MAP Quinasa Quinasa Quinasa 5/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Estrés Fisiológico/fisiología , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis/fisiología , Proteína 11 Similar a Bcl2 , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/fisiopatología , Proliferación Celular , Dietilnitrosamina/efectos adversos , Modelos Animales de Enfermedad , Hepatocitos/patología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/fisiopatología , MAP Quinasa Quinasa 4/fisiología , Masculino , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Lenvatinib is approved as a first-line treatment for unresectable HCC. The therapeutic duration of lenvatinib is limited by resistance, but the underlying mechanism is unclear. To establish lenvatinib-resistant cells, Hep3B cells were initially treated with 3 µM lenvatinib. The concentration was gradually increased by 1 µM or 0.5 µM per week and it reached to 7.5 µM 2 months after the initial exposure to lenvatinib. The biological characteristics of these cells were analyzed by ERK activation in the MAPK signaling pathway and a human phospho-receptor tyrosine kinase (RTK) antibody array. Factors possibly related to lenvatinib resistance were analyzed using inhibitors, and cell proliferation was analyzed. We established lenvatinib-resistant HCC cells (LR cells) by long-term exposure to lenvatinib. Lenvatinib reduced ERK activation in the parent cells, but not in the LR cells. RTK array analysis showed that the activities of EGFR and insulin-like growth factor 1 receptor (IGF1R)/insulin receptor (INSR) were significantly increased in LR cells, whereas the activities of other RTKs were unchanged. Erlotinib, a widely used EGFR inhibitor, downregulated ERK activation in LR cells. The proliferation of LR cells will also be affected when lenvatinib is combined with erlotinib to treat LR cells. In contrast, inhibition of IGFR/INSR did not affect ERK activation or cell proliferation. Scavenging of reactive oxygen species (ROS) ameliorated the enhanced EGFR activation in LR cells. Lenvatinib resistance was induced by enhanced EGFR activation, possibly via ROS accumulation, in lenvatinib- resistant cells. These findings may enable the development of lenvatinib combination therapies for HCC.
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Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Quinolinas , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Receptores ErbB/farmacología , Clorhidrato de Erlotinib/farmacología , Humanos , Neoplasias Hepáticas/patología , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinolinas/farmacología , Quinolinas/uso terapéutico , Especies Reactivas de Oxígeno/farmacologíaRESUMEN
BACKGROUND & AIMS: Mitogen-activated protein kinase (MAPK) signaling pathways regulate multiple cellular functions and are implicated in the pathogenesis of inflammatory bowel disease and colitis-associated cancer (CAC). Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase; little is known about the role of ASK1 in colonic disease. We assessed the involvement of ASK1 in the development of intestinal inflammation and CAC. METHODS: Dextran sodium sulfate (DSS) or Citrobacter rodentium was used to induce colitis in wild-type (WT) and ASK1 knock-out (ASK1(-/-)) mice; CAC was induced by azoxymethane injection followed by repeated intake of DSS by the mice. Primary macrophages were isolated from WT and ASK1(-/-) mice and used to investigate the involvement of ASK1 in innate immune responses. Bone marrow chimeric mice were used to study the contribution of myeloid cells to colitis activity. RESULTS: ASK1 deficiency increased susceptibility to colonic inflammation in both models of colitis. In vitro, ASK1(-/-) macrophages were impaired in their ability to kill bacteria and had increased susceptibility to bacterial-induced apoptosis, because p38 was inactivated. Expression of antiapoptotic genes was greatly reduced in ASK1(-/-) macrophages. WT mice given transplants of ASK1(-/-) mouse-derived bone marrow cells developed more severe DSS-induced colitis than mice with WT-derived bone marrow cells. In the CAC model, ASK1(-/-) mice developed more numerous and larger tumors than WT mice through increased colonic inflammation. CONCLUSIONS: ASK1 controls the development of intestinal inflammation and CAC through the regulation of innate immunity.
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Colitis/enzimología , Neoplasias del Colon/enzimología , Inmunidad Innata , MAP Quinasa Quinasa Quinasa 5/metabolismo , Macrófagos/enzimología , Animales , Apoptosis/genética , Azoximetano , Trasplante de Médula Ósea , Proliferación Celular , Células Cultivadas , Citrobacter rodentium , Colitis/inducido químicamente , Colitis/inmunología , Colitis/microbiología , Colitis/patología , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Femenino , Regulación de la Expresión Génica , Humanos , MAP Quinasa Quinasa Quinasa 5/deficiencia , MAP Quinasa Quinasa Quinasa 5/genética , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis , Interferencia de ARN , Índice de Severidad de la Enfermedad , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND & AIMS: Nuclear factor-kappaB (NF-kappaB) is an important transcription factor involved in various biological processes, including carcinogenesis. However, it is unknown whether NF-kappaB activation is involved in gastric carcinogenesis. METHODS: To explore the roles of inhibitor of kappaB kinase (IKKbeta), the key kinase for NF-kappaB activation, in gastric epithelium, we established a conditional gastric mucosal epithelium knockout mouse (Ikkbeta(DeltaST)). Gastric cancer was induced using N-methyl-N-nitrosourea (MNU). After 8 months, the number of tumors and their sizes were evaluated. Apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling staining, and levels of inflammatory cytokines were measured. RESULTS: No phenotypical or histologic difference was observed between untreated Ikkbeta(DeltaST) and controls (Ikkbeta(F/F)). The number of tumors was significantly less in the MNU-treated Ikkbeta(DeltaST) group than in the Ikkbeta(F/F) group (mean +/- standard error, 2.21 +/- 0.48 vs 0.80 +/- 0.23), and the size of the tumors did not differ (2.75 +/- 0.99 vs 2.89 +/- 1.12 mm). After a single oral dose of MNU, interleukin (IL)-1alpha was up-regulated significantly in control mice compared with Ikkbeta(DeltaST) mice, whereas the levels of IL-1beta, IL-6, and tumor necrosis factor-alpha were unchanged. MNU significantly increased apoptotic cell death in Ikkbeta(DeltaST) mice compared with Ikkbeta(F/F) mice, and apoptosis was dependent on decreased IL-1alpha expression. IL-1alpha also induced the proliferation of gastric cancer cells. Fewer tumors were observed in IL-1-receptor knockout mice (Il-1r(-/-); 1.17 +/- 0.44) than in control mice (2.42 +/- 0.52). CONCLUSIONS: IKKbeta regulates gastric carcinogenesis via IL-1alpha expression, which is associated with anti-apoptotic signaling and cell proliferation.
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Quinasa I-kappa B/fisiología , Interleucina-1alfa/fisiología , Neoplasias Gástricas/etiología , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Humanos , Interleucina-1alfa/genética , Metilnitrosourea/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/fisiología , Receptores de Interleucina-1/fisiología , Neoplasias Gástricas/patologíaRESUMEN
UNLABELLED: Nuclear factor kappaB (NF-kappaB) plays an important role in the regulation of innate immune responses, apoptosis, inflammation, and oncogenesis. NF-kappaB activation in the liver was observed after intrasplenic administration of a lung carcinoma cell line, LLC, which induces liver metastasis. To explore the role of Ikappa B kinase beta (IKKbeta), which is the critical kinase of the IKK complex, and NF-kappaB activation in metastasis, we injected LLC cells into hepatocyte-specific IKKbeta knockout mice (Ikkbeta(Deltahep)), whole-liver knockout (Ikkbeta(DeltaL+H)) mice, and control (Ikkbeta(F/F)) mice. Ikkbeta(DeltaL+H) mice developed liver metastasis with significantly lower liver weights and fewer metastatic foci compared to Ikkbeta(Deltahep) and Ikkbeta(F/F) mice. Furthermore, intrasplenic LLC injection induced the messenger RNA (mRNA) expression of interleukin (IL)-6 and IL-1beta in Ikkbeta(F/F) mice, whereas these genes were less expressed in Ikkbeta(DeltaL+H) mice. IL-6(-/-) mice and treatment with anti-IL-6 receptor antibody showed a lesser degree of metastatic tumor, indicating that IL-6 is associated with liver metastasis. CONCLUSION: Collectively, these observations suggest that IKKbeta/NF-kappaB activation controls the development of liver metastasis by way of IL-6 expression and is a potential target for the development of antimetastatic drugs.
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Quinasa I-kappa B/fisiología , Interleucina-6/fisiología , Neoplasias Hepáticas/secundario , FN-kappa B/fisiología , Animales , Proliferación Celular , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/etiología , Péptidos/farmacología , Factor de Necrosis Tumoral alfa/fisiología , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
PURPOSE: Nuclear factor kappaB (NF-kappaB) is an important transcription factor in various biological processes. Constitutive NF-kappaB activation has been noted in many tumors, including colorectal cancers. However, the precise role of this activation in colorectal cancer is unclear. EXPERIMENTAL DESIGN: Constitutive NF-kappaB activation was evaluated in colorectal cancer tissues and cell lines. To inhibit NF-kappaB activation, we established cancer cells with stable knockdown of IkappaB kinase gamma (NF-kappaB essential modulator), which is the regulatory subunit of the IkappaB kinase complex, by RNA interference. Cell growth and apoptosis were evaluated in wild-type cells (WT) and knocked-down cells (KD). Microarray and protein array analysis were also done. To determine involvement of angiogenesis, human umbilical vein endothelial cells were used. By s.c. transplantation of the cells into nude mice, tumor sizes, vascularity, and chemodrug sensitivity were analyzed. RESULTS: Constitutive NF-kappaB activation was observed in 40% of colorectal cancer tissues and 67% of cell lines. Cell proliferation was not different between WT and KD in vitro, whereas apoptosis mediated by tumor necrosis factor-alpha and 5-fluorouracil were increased in KD. Several angiogenic chemokines were decreased in KD. Human umbilical vein endothelial cells incubated in WT supernatant showed more branch points than in KD, suggesting that constitutive NF-kappaB activation was involved in angiogenesis. Subcutaneous tumor expansion was suppressed to 23% in KD, and vessels were also decreased. By 5-fluoruracil treatment, tumor expansion was suppressed to a greater extent in KD (to 6%) than in WT (to 50%). CONCLUSION: NF-kappaB inhibition may represent a potent treatment modality in colorectal cancer, especially in cases with constitutive NF-kappaB activation.
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Inductores de la Angiogénesis/metabolismo , Carcinoma/metabolismo , Neoplasias Colorrectales/metabolismo , FN-kappa B/metabolismo , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis , Carcinoma/irrigación sanguínea , Carcinoma/patología , Línea Celular , Línea Celular Tumoral , Quimiocinas/metabolismo , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/patología , Fluorouracilo/farmacología , Humanos , Quinasa I-kappa B/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones , Ratones Desnudos , Neovascularización Patológica/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND & AIMS: E-cadherin (Cdh1) is a key molecule for adherence required for maintenance of structural homeostasis. Loss of E-cadherin leads to poor prognosis and the development of resistance to chemotherapy in pancreatic cancer. Here, we evaluated the physiological and pathologic roles of E-cadherin in the pancreas. METHODS: We crossbred Ptf1a-Cre mice with Cdh1f/f mice to examine the physiological roles of E-cadherin in the pancreas. In addition, we crossbred these mice with LSL-KrasG12D/+ mice (PKC) to investigate the pathologic roles of E-cadherin. We also generated a tamoxifen-inducible system (Ptf1a-CreERT model). Organoids derived from these models using lentiviral transduction were analyzed for immunohistochemical features. Established cell lines from these organoids were analyzed for migratory and invasive activities as well as gene expression by complementary DNA microarray analyses. RESULTS: None of the Ptf1a-Cre mice crossbred with Cdh1f/f mice survived for more than 28 days. We observed aberrant epithelial tubules that resembled the structure of acinar-to-ductal metaplasia after postnatal day 6, showing features of pancreatitis. All of the PKC mice died within 10 days. We observed tumorigenicity with increasing stroma-like aggressive tumors. Ptf1a-CreERT models showed that deletion of E-cadherin led to earlier pancreatic intraepithelial neoplasm formation. Cells established from PKC organoids had greater migratory and invasive activities, and these allograft tumors showed a poorly differentiated phenotype. Gene expression analysis indicated that Hdac1 was up-regulated in PKC cell lines and a histone deacetylase 1 inhibitor suppressed PKC cell proliferation. CONCLUSIONS: Under physiological conditions, E-cadherin is important for maintaining the tissue homeostasis of the pancreas. Under pathologic conditions with mutational Kras activation, E-cadherin plays an important role in tumor formation via the acquisition of tumorigenic activity.