RESUMEN
To explore the mechanism of coadaptation and the potential drivers of pancreatic ductal adenocarcinoma (PDAC) metastasis to the liver, we study key molecules involved in this process and their translational value. Premetastatic niche (PMN) and macrometastatic niche (MMN) formation in a mouse model is observed via CT combined with 3D organ reconstruction bioluminescence imaging, and then we screen slit guidance ligand 2 (SLIT2) and its receptor roundabout guidance receptor 1 (ROBO1) as important factors. After we confirm the expression and distribution of SLIT2 and ROBO1 in samples from PDAC patients and several mouse models, we discover that SLIT2-ROBO1-mediated coadaptation facilitated the implantation and outgrowth of PDAC disseminated tumour cells (DTCs) in the liver. We also demonstrate the dependence receptor (DR) characteristics of ROBO1 in a follow-up mechanistic study. A neutralizing antibody targeting ROBO1 significantly attenuate liver metastasis of PDAC by preventing the coadaptation effect. Thus, we demonstrate that coadaptation is supported by the DR characteristics in the PMN and MMN.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Hepáticas , Neoplasias Pancreáticas , Animales , Ratones , Carcinoma Ductal Pancreático/genética , Movimiento Celular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Transducción de Señal , Neoplasias PancreáticasRESUMEN
The development of resistance to anticancer drugs is believed to cause chemotherapy failure in pancreatic cancer (PC). The efflux of anticancer drugs mediated by ATP-binding cassette (ABC) transporters is a widely accepted mechanism for chemoresistance, but for ABCA subfamily members, which are characterized by their ability to transport lipids and cholesterol, its role in chemoresistance remains unknown. Here we found that the expression of ABCA8, a member of ABCA subfamily transporters, was significantly increased in human PC cells after gemcitabine (GEM) treatment, as well as in established GEM-resistant (Gem-R) PC cells. Importantly, ABCA8 knockdown reversed the chemoresistance phenotype of Gem-R cells, whereas ABCA8 overexpression significantly decreased the sensitivity of human PC cells to GEM, both in vitro and in vivo, demonstrating an important role of ABCA8 in regulating chemosensitivity. Moreover, our results showed that treatment with taurocholic acid (TCA), an endogenous substrate of ABCA8, also induced GEM insensitivity in PC cells. We further demonstrated that ABCA8 mediates the efflux of TCA out of PC cells, and that extracellular TCA activates extracellular signal-regulated kinase (ERK) signaling via the sphingosine 1-phosphate receptor 2 (S1PR2), which is responsible for ABCA8-induced GEM ineffectiveness. Together, these findings reveal a novel TCA-related mechanism of ABCA subfamily transporter-mediated chemoresistance that goes beyond the role of a drug pump and suggest ABCA8 or the TCA-S1RP2-ERK pathway as potential targets for improving the effectiveness of and overcoming the resistance to chemotherapy in PC.
RESUMEN
The overarching view of current tumor therapies simplifies cancer to a cell-biology problem in which neoplasms are caused solely by malignant cells and the exploration of carcinogenesis and tumor progression largely focuses on somatic mutations and other genetic abnormalities of cancer cells. The limited therapeutic response indicates that cancer is driven not only by endogenous oncogenic factors and reciprocal interactions within the tumor microenvironment, but also by complex systemic processes. Homeostasis is the fundamental premise of health, and is maintained by systemic regulation of neuro-endocrine-immune axis. Cancer is also a systemic disease that manifested by dysfunction of the nervous, endocrine, and immune systems. Multiple axes of regulation exist in cancer, including central-, organ-, and microenvironment-level manipulation. At each specific regulatory level, the tridirectional communication among the nervous, endocrine, and immune factors transmit flexible signaling to induce proliferation, invasion, reprogrammed metabolism, therapeutic resistance, and other malignant phenotypes of cancer cells, resulting in the extremely poor prognosis of this lethal disease. Understanding this coordinated signaling network will enable the development of new approaches for cancer treatment via behavioral and pharmacological interventions.
RESUMEN
Accumulating evidence has pointed out that metastasis is the leading cause of death in several malignant tumor, including CRC. During CRC, metastatic capacity is closely correlated with reprogrammed energy metabolism. Mitochondrial Pyruvate Carrier 1 (MPC1), as the carrier of transporting pyruvate into mitochondria, linked the glycolysis and TCA cycle, which would affect the energy production. However, the specific role of MPC1 on tumor metastasis in CRC remains unexplored. Here, by data mining of genes involved in pyruvate metabolism using the TCGA dataset, we found that MPC1 was significantly downregulated in CRC compared to nontumor tissues. Similar MPC1 expression pattern was also found in multiple GEO datasets. IHC staining in both human sample and AOM/DSS induced mouse CRC model revealed significant downregulation of MPC1. What is more, we found that MPC1 expression was gradually decreased in normal tissue, primary CRC, and metastasis CRC. Additionally, poor prognosis emerged in the MPC1 low expression patients, especially in patients with metastasis. Following, functional tests showed that MPC1 overexpression inhibited the motility of CRC cells in vitro and MPC1 silencing enhanced liver metastases in vivo. Furthermore, we uncovered that decreased MPC1 activated the Wnt/ß-catenin pathway by promoting nuclear translocation of ß-catenin to mediate the expression of MMP7, E-cadherin, Snail1, and myc. Collectively, our data suggest that MPC1 has the potential to be served as a promising biomarker for diagnosis and a therapeutic target in CRC.
Asunto(s)
Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/metabolismo , Neoplasias Hepáticas/secundario , Proteínas de Transporte de Membrana Mitocondrial/deficiencia , Transportadores de Ácidos Monocarboxílicos/deficiencia , beta Catenina/metabolismo , Adulto , Anciano , Biomarcadores , Línea Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Bases de Datos Genéticas , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Transporte de Proteínas , Carga Tumoral , Vía de Señalización WntRESUMEN
BACKGROUND: Collagens are the most abundant proteins in extra cellular matrix and important components of tumor microenvironment. Recent studies have showed that aberrant expression of collagens can influence tumor cell behaviors. However, their roles in hepatocellular carcinoma (HCC) are poorly understood. METHODS: In this study, we screened all 44 collagen members in HCC using whole transcriptome sequencing data from the public datasets, and collagen type IV alpha1 chain (COL4A1) was identified as most significantly differential expressed gene. Expression of COL4A1 was detected in HCC samples by quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry (IHC). Finally, functions and potential mechanisms of COL4A1 were explored in HCC progression. RESULTS: COL4A1 is the most significantly overexpressed collagen gene in HCC. Upregulation of COL4A1 facilitates the proliferation, migration and invasion of HCC cells through FAK-Src signaling. Expression of COL4A1 is upregulated by RUNX1 in HCC. HCC cells with high COL4A1 expression are sensitive to the treatment with FAK or Src inhibitor. CONCLUSION: COL4A1 facilitates growth and metastasis in HCC via activation of FAK-Src signaling. High level of COL4A1 may be a potential biomarker for diagnosis and treatment with FAK or Src inhibitor for HCC.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/secundario , Colágeno Tipo IV/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/patología , Familia-src Quinasas/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Estudios de Casos y Controles , Movimiento Celular , Proliferación Celular , Colágeno Tipo IV/genética , Femenino , Quinasa 1 de Adhesión Focal/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Desnudos , Invasividad Neoplásica , Fosforilación , Pronóstico , Transducción de Señal , Células Tumorales Cultivadas , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Familia-src Quinasas/genéticaRESUMEN
Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a malignant disease with a poor prognosis. One prominent aspect of PDAC that contributes to its aggressive behavior is its altered cellular metabolism. The aim of this study was to characterize the oncogenic effects of ubiquinol-cytochrome c reductase core protein I (UQCRC1), a key component of mitochondrial complex III, in PDAC development and to assess its potential as a therapeutic target for PDAC. Experimental Design: The expression of UQCRC1 in human PDAC tissues and p48-Cre/p53Flox/WT/LSL-KrasG12D (KPC) mouse pancreatic intraepithelial neoplasias (PanINs) was determined by immunohistochemistry. The role of UQCRC1 in promoting PDAC growth was evaluated in vitro in PANC-1 and CFPAC-1 cells and in vivo in transplanted mouse models of PDAC. Extracellular flux and RNA-Seq analyses were applied to investigate the mechanism of UQCRC1 in the regulation of mitochondrial metabolism and PDAC cell growth. The therapeutic potential of UQCRC1 in PDAC was assessed by knockdown of UQCRC1 using an RNA interference approach. Results: UQCRC1 expression showed a gradual increase during the progression from PanIN stages to PDAC in KPC mice. Elevated expression of UQCRC1 was observed in 72.3% of PDAC cases and was correlated with poor prognosis of the disease. UQCRC1 promoted PDAC cell growth in both in vitro experiments and in vivo subcutaneous and orthotopic mouse models. UQCRC1 overexpression resulted in increased mitochondrial oxidative phosphorylation (OXPHOS) and ATP production. The overproduced ATP was released into the extracellular space via the pannexin 1 channel and then functioned as an autocrine or paracrine agent to promote cell proliferation through the ATP/P2Y2-RTK/AKT axis. UQCRC1 knockdown or ATP release blockage could effectively inhibit PDAC growth. Conclusion: UQCRC1 has a protumor function and may serve as a potential prognostic marker and therapeutic target for PDAC.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Complejo III de Transporte de Electrones/genética , Proteínas Mitocondriales/metabolismo , Neoplasias Pancreáticas/patología , Adenosina Trifosfato/metabolismo , Anciano , Animales , Carcinogénesis/genética , Carcinoma in Situ/metabolismo , Carcinoma Ductal Pancreático/terapia , Línea Celular Tumoral/trasplante , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Persona de Mediana Edad , Fosforilación Oxidativa , Interferencia de ARN/efectos de los fármacosRESUMEN
BACKGROUND: Liver is one of the most preferred destinations of distant metastasis in gastric cancer (GC). As effective treatment is still limited, the prognosis of GC patients bearing liver metastasis is poor. We filter out lysyl oxidase (LOX) to study its function in the tumor microenvironment (TME) and seek for potential therapeutic targets. METHODS: Transcription analysis on 6 cases of liver metastasis of GC patients with respective paired primary tumors and adjacent normal livers was performed. The filtration out of LOX was done using 5 datasets. 69 GC liver metastasis tissues were utilized to perform immunohistochemistry (IHC) and analyze prognosis. Computed Tomography (CT) combined 3D organ reconstruction bioluminescence imaging was performed to precisely evaluate the metastatic tumor burden on liver of intrasplenic injection mouse model. Human and mouse cancer associated fibroblasts (CAFs) in liver metastasis were separated to culture to study the interaction of LOX and TGF-ß1. Patients-derived xenograft (PDX) model was established using liver metastasis of patients to evaluate the therapeutic value of LOX inhibitor ß-aminopropionitrile (BAPN). RESULTS: CAFs-derived LOX at liver metastatic niche of GC promotes niche formation and outgrowth thus predicts poor prognosis. Meanwhile tumor cells in niche secrete TGF-ß1 to nourish CAFs and stimulate them to produce more LOX in turn. The mechanism involved in LOX-mediated proliferation facilitation is enhancement of Warburg effect. The inhibitor of LOX, BPAN could hamper the effect brought by LOX in vivo and in vitro. INTERPRETATION: Our study has unveiled a positive feedback loop between CAFs and tumor cells in liver metastasis niche of GC. The core molecule is LOX which facilitates Warburg effect. Targeting LOX with its inhibitor BAPN might serve as a potential therapeutic strategy. FUND: This research was supported by the National Natural Science Foundation of China (31872740), the 100-member plan of the Shanghai Municipal Commission of Health and Family Planning (2017BR043), Shanghai Science and Technology Commission Project(17ZR1416800), Renji Hospital Training Fund (PYMDT-003, PYIII-17-015), National Natural Science Foundation of China (81672358), the Shanghai Municipal Education Commission-Gao feng Clinical MedicineGrant Support (20181708), Program of Shanghai Academic/Technology Research Leader(19XD1403400), Science and Technology Commission of Shanghai Municipality (18410721000), Shanghai Municipal Health Bureau (2018BR32), China Postdoctoral Science Foundation (2018M640403), National Natural Science Foundation of China (81701945) and Youth project of Shanghai Municipal Health Commission(20164Y0045).
Asunto(s)
Fibroblastos Asociados al Cáncer/enzimología , Fibroblastos Asociados al Cáncer/patología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/secundario , Proteína-Lisina 6-Oxidasa/metabolismo , Neoplasias Gástricas/patología , Aminopropionitrilo/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular , Glucólisis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones Endogámicos C57BL , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Células del Estroma/patología , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia ArribaRESUMEN
Acetyl-CoA synthetase 2 (ACSS2) generates acetyl-CoA from acetate is important for histone acetylation and gene expression. ACSS2 fulfills distinct functions depending on its cellular location in tumor cells. The role and cellular localization of ACSS2 in hepatocellular carcinoma (HCC) remains to be studied. Herein, we identified that the alternative transcription start site selection of ACSS2 was significantly different between HCC and corresponding adjacent tissues. Alternative transcription start site selection produced two different ACSS2 transcripts, ACSS2-S1 and ACSS2-S2. The two isoforms of ACSS2 had different subcellular localization and different functions. Overexpression of ACSS2-S2 promoted cell proliferation and invasion, but ACSS2-S1 did not. The ACSS2-S1 was mainly present in cytoplasm, and ACSS2-S2 was distributed in both nucleus and cytoplasm. Finally, we demonstrated that alternative transcription start site selection of ACSS2 correlates ribosome biogenesis in HCC. Our findings reveal an oncogenic role of ACSS2-S2 in HCC progression via increase of ribosome biogenesis, and suggest ACSS2-S2 might be a potential therapeutic target against the HCC.
Asunto(s)
Acetato CoA Ligasa/genética , Carcinoma Hepatocelular/metabolismo , Núcleo Celular/metabolismo , Neoplasias Hepáticas/metabolismo , Ribosomas/metabolismo , Sitio de Iniciación de la Transcripción , Acetato CoA Ligasa/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Invasividad Neoplásica , Pronóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ribosomas/genéticaRESUMEN
BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death worldwide. Neurotransmitter-initiated signalling pathway is profoundly implicated in tumour initiation and progression. Here, we investigated whether dysregulated neurotransmitter receptors play a role during pancreatic tumourigenesis. METHODS: The Cancer Genome Atlas and Gene Expression Omnibus datasets were used to identify differentially expressed neurotransmitter receptors. The expression pattern of gamma-aminobutyric acid type A receptor pi subunit (GABRP) in human and mouse PDAC tissues and cells was studied by immunohistochemistry and western blot analysis. The in vivo implications of GABRP in PDAC were tested by subcutaneous xenograft model and lung metastasis model. Bioinformatics analysis, transwell experiment and orthotopic xenograft model were used to identify the in vitro and in vivo effects of GABRP on macrophages in PDAC. ELISA, co-immunoprecipitation, proximity ligation assay, electrophysiology, promoter luciferase activity and quantitative real-time PCR analyses were used to identify molecular mechanism. RESULTS: GABRP expression was remarkably increased in PDAC tissues and associated with poor prognosis, contributed to tumour growth and metastasis. GABRP was correlated with macrophage infiltration in PDAC and pharmacological deletion of macrophages largely abrogated the oncogenic functions of GABRP in PDAC. Mechanistically, GABRP interacted with KCNN4 to induce Ca2+ entry, which leads to activation of nuclear factor κB signalling and ultimately facilitates macrophage infiltration by inducing CXCL5 and CCL20 expression. CONCLUSIONS: Overexpressed GABRP exhibits an immunomodulatory role in PDAC in a neurotransmitter-independent manner. Targeting GABRP or its interaction partner KCNN4 may be an effective therapeutic strategy for PDAC.
Asunto(s)
Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Receptores de GABA-A/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Macrófagos/fisiología , Ratones , Transducción de Señal/fisiologíaRESUMEN
Long noncoding RNAs (lncRNAs) are implicated as novel drivers in hepatocellular carcinoma (HCC), but the underlying mechanisms of this relationship with hepatocarcinogenesis are unknown. We report a novel, liver-specific lncRNA LINC01093 that shows significant downregulation in HCC tissues. LINC01093 expression is inversely correlated with cancer embolus and HCC TNM stage and as a prognostic predictor for HCC patients. LINC01093 overexpression significantly suppresses HCC cell proliferation and metastasis in vitro and in vivo. Conversely, its knockdown promotes HCC progression. Mechanistic analyses indicate that LINC01093 directly binds insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), interfering with interaction between IGF2BP1 and glioma-associated oncogene homolog 1 (GLI1) mRNA. The result is degradation of GLI1 mRNA, further affecting expression of GLI1 downstream molecules involved in HCC progression. The liver-enriched lncRNA LINC01093 is a promising prognostic indicator for HCC patients, and the newly identified LINC01093-IGF2BP1-GLI1 axis shows potential for therapeutic targets in HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteína con Dedos de Zinc GLI1/genética , Animales , Carcinogénesis , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Regulación hacia Abajo , Genes Supresores de Tumor , Xenoinjertos , Humanos , Neoplasias Hepáticas/metabolismo , Ratones , Metástasis de la Neoplasia , ARN Largo no Codificante/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
Adipokines are emerging as a link between obesity and obesity-related cancers, including pancreatic cancer. Adiponectin is an abundant adipokine with pleiotropic beneficial roles in metabolic disorders. Low adiponectin levels are commonly observed in human obesity and have been associated with increased pancreatic cancer risk in prospective epidemiologic studies. Here, we investigated the direct effect of adiponectin on human pancreatic cancer in vitro and in vivo. Our results showed that adiponectin treatment significantly inhibited the proliferation of human pancreatic cancer cells. Knockdown of adiponectin receptors completely eliminated the antiproliferation effect of adiponectin and markedly promoted the growth of human pancreatic cancer xenografts in nude mice. Further analysis revealed that adiponectin blocked the phosphorylation/inactivation of GSK-3ß, suppressed the intracellular accumulation of ß-catenin, reduced the expression of cyclin D1, and consequently caused cell cycle accumulation at the G0-G1 phase in pancreatic cancer cells. Adiponectin-mediated attenuation of cell proliferation was abrogated by the GSK-3ß inhibitor. In addition, a microarray analysis revealed that adiponectin also downregulated the expression of TCF7L2, a coactivator of ß-catenin, at the transcriptional level in pancreatic cancer cells. These results indicated that the protective role of adiponectin against human pancreatic cancer might be attributed to its attenuating effect on the ß-catenin signaling pathway. Taken together, our findings support a causal link between hypoadiponectinemia and increased pancreatic cancer risk, and suggest that activating adiponectin signaling could be a novel therapeutic strategy for obesity-related pancreatic cancer.
Asunto(s)
Adiponectina/farmacología , Neoplasias Pancreáticas/metabolismo , Receptores de Adiponectina/metabolismo , beta Catenina/metabolismo , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Biología Computacional , Ciclina D1/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Neoplasias Pancreáticas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Adiponectina/genética , Proteína 2 Similar al Factor de Transcripción 7/genética , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Hepatic fibrosis is caused by chronic liver injury and may progress toward liver cirrhosis, and even hepatocellular carcinoma. However, current treatment is not satisfactory. Therefore, there is a mandate to find novel therapeutic targets to improve therapy, and biomarkers to monitor therapeutic response. METHODS: Liver fibrosis was induced by carbon tetrachloride (CCl4) or thioacetamide (TAA) in wild type (WT) or CTHRC1-/- mice, followed by immunofluorescence and immunohistochemical analyses. CTHRC1 monoclonal antibody (mAb) was used to abrogate the effect of CTHRC1 in vitro and in vivo. RESULTS: Here, we reported that collagen triple helix repeat containing 1 (CTHRC1), a secreted protein derived from hepatic stellate cells (HSCs), was significantly up-regulated in fibrotic liver tissues. CTHRC1 promoted HSCs transformation from a quiescent to an activated state, and enhanced migratory or contractile capacities of HSCs by activating TGF-ß signaling. Meanwhile, CTHRC1 competitively bound to Wnt noncononical receptor and promoted the contractility but not activation of HSCs. CCl4 or TAA-induced liver fibrosis was attenuated in CTHRC-/- mice compared with littermate control, while a monoclonal antibody of CTHRC1 suppressed liver fibrosis in WT mice treated with CCl4 or TAA. INTERPRETATION: We demonstrated that CTHRC1 is a new regulator of liver fibrosis by modulating TGF-ß signaling. Targeting CTHRC1 could be a promising therapeutic approach, which can suppress TGF-ß signaling and avoid the side effects caused by directly targeting TGF-ß. CTHRC1 could also be a potential biomarker for monitoring response to anti-fibrotic therapy. FUND: This study was supported by the National Natural Science Foundation of China (ID 81672358, 81871923, 81872242, 81802890), the Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (ID 20181708), the Natural Science Foundation of Shanghai (ID 17ZR1428300, 18ZR1436900), and Shanghai Municipal Health Bureau (ID 2018BR32). The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript.
Asunto(s)
Comunicación Autocrina , Proteínas de la Matriz Extracelular/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular , Movimiento Celular , Células Cultivadas , Colágeno/química , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/química , Humanos , Cirrosis Hepática/patología , Masculino , Ratones , Ratones Noqueados , Modelos Biológicos , Ratas , Vía de Señalización WntRESUMEN
The maturation of natural killer (NK) cells is critical for the acquisition of robust effector functions and the immune response to tumors. However, the influence of psychological stress on NK-cell maturation remains unknown. In this study, we investigated the alteration of NK-cell maturation in response to enriched environment (EE) exposure, which induced eustress, or positive stress, in mice. Analysis of markers representing distinct mature stages revealed that EE promoted the terminal maturation of NK cells both centrally in the bone marrow and peripherally in the spleen and blood. Additionally, EE increased CD27+ immature and intermediate-mature NK cell proliferation in the bone marrow. Furthermore, EE exposure brought about a similar promoting effect on NK-cell maturation in tumor-bearing mice. In tumor-bearing mice, EE substantially enhanced the proliferative potential of splenic CD27+ NK cells compared to those in the bone marrow. EE-housed mice displayed a tumor-resistant phenotype and an increased proportion of intratumoral NK cells, especially CD11b+ CD27- mature NK cells, while splenectomy abolished the tumor-retardant effect caused by EE and EE-induced NK-cell infiltration into tumors. Given that our previous study demonstrated an important role for NK cells in EE-induced tumor inhibition, the findings of this study further indicate that the enhanced maturation and proliferation of splenic NK cells may contribute to EE-induced tumor inhibition to some extent. Taken together, the results of this study suggest a positive modulating effect of environment-induced eustress on NK-cell maturation, with potential implications for understanding how eustress boosts NK-cell antitumor immunity.
Asunto(s)
Ambiente , Células Asesinas Naturales/inmunología , Estrés Psicológico/inmunología , Animales , Médula Ósea , Células de la Médula Ósea/inmunología , Diferenciación Celular/inmunología , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Citotoxicidad Inmunológica/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/fisiología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , Bazo/inmunologíaRESUMEN
BACKGROUND/AIMS: Epidermal growth factor receptor variant III (EGFRvIII), the most frequent EGFR variant, is constitutively activated without binding to EGF and is correlated with a poor prognosis. CH12, a human-mouse chimeric monoclonal antibody, has been developed in our laboratory and selectively binds to overexpressed EGFR and EGFRvIII. A previous study had reported that EGFR could influence autophagic activity, and autophagy is closely related to tumor development and the response to drug therapy. In this study, we aimed to elucidate the effect of CH12 on autophagy and efficacy of combining CH12 with an autophagy inhibitor against EGFRvIII-positive tumors. METHODS: EGFRvIII was overexpressed in liver cancer, glioblastoma and breast cancer, and the change in the autophagy-relevant protein levels was analyzed by western blot assays, LC3 punctate aggregation was analyzed by immunofluorescence. The interaction of Beclin-1 and Rubicon was assessed by co-immunoprecipitation (Co-IP) after CH12 treatment. The efficacy of ATG7 or Beclin-1 siRNA in combination with CH12 in Huh-7-EGFRvIII cells was assessed by CCK-8 assays. The autophagy and apoptosis signaling events in Huh-7-EGFRvIII cells upon treatment with control, CH12, siRNA or combination for 48 h were assessed by western blot assays. RESULTS: Our results showed that, in cancer cell lines overexpressing EGFRvIII, only the liver cancer cell lines Huh-7 and PLC/PRF/5 suggested autophagy activation. We then investigated the mechanism of autophagy activation after EGFRvIII overexpression. The results showed that EGFRvIII interacted with Rubicon, an autophagy inhibition protein, and released Beclin-1 to form the inducer complex, thus contributing to autophagy. In addition, CH12, via inhibiting the phosphorylation of EGFRvIII, promoted the interaction of EGFRvIII with Rubicon, further inducing autophagy. In vitro assays suggested that knocking down the expression of the key proteins ATG7 or Beclin-1 in the autophagy pathway with siRNA inhibits tumor cell proliferation. Combining autophagy-related proteins 7 (ATG7) or Beclin-1 siRNA with CH12 in Huh-7-EGFRvIII cells showed better inhibition of cell proliferation. CONCLUSION: EGFRvIII could induce autophagy, and CH12 treatment could improve autophagy activity in EGFRvIII-positive liver cancer cells. The combination of CH12 with an autophagy inhibitor or siRNA against key proteins in the autophagy pathway displayed more significant efficacy on EGFRvIII-positive tumor cells than monotherapy, and induced cell apoptosis.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Autofagia/efectos de los fármacos , Receptores ErbB/inmunología , Anticuerpos Monoclonales/inmunología , Proteína 7 Relacionada con la Autofagia/antagonistas & inhibidores , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia , Beclina-1/antagonistas & inhibidores , Beclina-1/genética , Beclina-1/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptores ErbB/genética , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Células MCF-7 , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Fosforilación/efectos de los fármacos , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína Sequestosoma-1/metabolismoRESUMEN
Dysregulated potassium (K+) channels have previously been shown to promote the development and progression of many types of cancers. Meanwhile, K+ channels are particularly important in regulating the endocrine and exocrine functions of pancreas. However, the expression pattern and prognostic significance of K+ channels in pancreatic ductal adenocarcinoma (PDAC) remain unknown. In this study, by screening a GEO dataset containing 36 microdissected PDAC and matching normal pancreatic tissue samples, four differentially expressed K+ channels (KCNJ5, KCNJ16, KCNN4 and KCNK1) were identified in PDAC. by immunohistochemical analysis of pancreatic tissue sections from Pdx1-Cre; LSL-KrasG12D/+ mice (KC), Pdx1-Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+ mice (KPC) and human PDAC tissue microarrays, we found that Ca2+-activated K+ channel KCNN4 was significantly elevated in pancreatic intraepithelial neoplasia (PanIN) and PDAC epithelia compared with untransformed pancreas tissues. Higher epithelial KCNN4 expression was closely correlated with advanced TNM stages and predicted a poor prognosis in patients with PDAC. Elevated KCNN4 expression was significantly associated with shorter survival in univariable and multivariable analyses. Collectively, the identification of expression pattern of K+ channels in PDAC and its precursor PanIN demonstrates the importance of KCNN4 channel during the malignant transformation of PDAC. On the basis of the prognostic signals from two independent cohorts, KCNN4 should be considered as a promising therapeutic target.
Asunto(s)
Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Anciano , Animales , Biomarcadores de Tumor/metabolismo , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patología , Carcinoma Ductal Pancreático/patología , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Neoplasias Pancreáticas/patología , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , PronósticoRESUMEN
The incorporation of an endogenous safety switch represents a rational strategy for the control of toxicities following the administration of adoptive T cell therapies. An ideal safety switch should be capable of depleting the transferred T cells with minimal injury to normal tissues. We generated a fusion receptor by engineering a cryptic 806 epitope of human epidermal growth factor receptor (EGFR) into the N terminus of the full-length human folate receptor 1 (FOLR1), designated as FR806. The expression of FR806 allows transduced T cells to be targeted with CH12, a monoclonal antibody recognizing the 806 epitope, but not wild-type EGFR in healthy tissues. FR806, therefore, constitutes a specific cell-surface marker for the elimination of transduced T cells. We demonstrate that the antibody-drug conjugate (ADC) CH12-MMAF is efficiently internalized by FR806-expressing T cells and has the potential to eliminate them. Transfected T cells could, furthermore, be efficiently detected and purified using CH12 antibodies. In immuno-compromised mice, CH12-MMAF eliminated the majority of transferred T cells expressing FR806 and anti-CD19 chimeric antigen receptor (CAR). The selectivity for the 806 epitope and internalization capacity of FOLR1 makes FR806 an efficient safety switch, which may additionally be used as a detection and purification biomarker for human T cell immunotherapies.
Asunto(s)
Traslado Adoptivo/métodos , Biomarcadores/sangre , Linfocitos T/inmunología , Animales , Línea Celular , Humanos , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Ratones , Ratones SCID , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND & AIMS: Individuals with Down syndrome have a low risk for many solid tumors, prompting the search for tumor suppressor genes on human chromosome 21 (HSA21). We aimed to identify and explore potential mechanisms of tumor suppressors on HSA21 in hepatocellular carcinoma (HCC). METHODS: We compared expression of HSA21 genes in 14 pairs of primary HCC and adjacent noncancer liver tissues using the Affymetrix HG-U133 Plus 2.0 array (Affymetrix, Santa Clara, CA). HCC tissues and adjacent normal liver tissues were collected from 108 patients at a hospital in China for real-time polymerase chain reaction and immunohistochemical analyses; expression levels of regulator of calcineurin 1 (RCAN1) isoform 4 (RCAN1.4) were associated with clinical features. We overexpressed RCAN1.4 from lentiviral vectors in MHCC97H and HCCLM3 cells and knocked expression down using small interfering RNAs in SMMC7721 and Huh7 cells. Cells were analyzed in proliferation, migration, and invasion assays. HCC cells that overexpressed RCAN1.4 or with RCAN1.4 knockdown were injected into livers or tail veins of nude mice; tumor growth and numbers of lung metastases were quantified. We performed bisulfite pyrosequencing and methylation-specific polymerase chain reaction analyses to analyze CpG island methylation. We measured phosphatase activity of calcineurin in HCC cells. RESULTS: RCAN1.4 mRNA and protein levels were significantly decreased in primary HCC compared with adjacent noncancer liver tissues. Reduced levels of RCAN1.4 mRNA were significantly associated with advanced tumor stages, poor differentiation, larger tumor size, and vascular invasion. Kaplan-Meier survival analysis showed that patients with HCCs with lower levels of RCAN1.4 mRNA had shorter time of overall survival and time to recurrence than patients whose tumors had high levels of RCAN1.4 mRNA. In HCC cell lines, expression of RCAN1.4 significantly reduced proliferation, migration, and invasive activity. HCC cells that overexpressed RCAN1.4 formed smaller xenograft tumors, with fewer metastases and blood vessels, than control HCC cells. In HCC cells, RCAN1.4 inhibited expression of insulin-like growth factor 1 and vascular endothelial growth factor A by reducing calcineurin activity and blocking nuclear translocation of nuclear factor of activated T cells (NFAT1). HCC cells incubated with the calcineurin inhibitor cyclosporin A had decreased nuclear level of NFAT1. HCC cells had hypermethylation of a CpG island in the 5' regulatory region of RCAN1.4, which reduced its expression. CONCLUSIONS: RCAN1.4 is down-regulated in HCC tissues, compared with non-tumor liver tissues. RCAN1.4 prevents cell proliferation, migration, and invasion in vitro; overexpressed RCAN1.4 in HCC cells prevents growth, angiogenesis, and metastases of xenograft tumors by inhibiting calcineurin activity and nuclear translocation of NFAT1.
Asunto(s)
Calcineurina/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Proteínas Musculares/genética , Factores de Transcripción NFATC/metabolismo , ARN Mensajero/análisis , Adulto , Anciano , Animales , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/secundario , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Cromosomas Humanos Par 21 , Islas de CpG/genética , Metilación de ADN , Proteínas de Unión al ADN , Supervivencia sin Enfermedad , Regulación hacia Abajo , Femenino , Expresión Génica , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/análisis , Hígado/química , Neoplasias Hepáticas/química , Masculino , Ratones , Persona de Mediana Edad , Proteínas Musculares/análisis , Factores de Transcripción NFATC/genética , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Trasplante de Neoplasias , Isoformas de Proteínas/genética , Transporte de Proteínas/efectos de los fármacos , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Tasa de Supervivencia , Carga Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND & AIMS: Desmoplasia and poor vascularity cause severe metabolic stress in pancreatic ductal adenocarcinomas (PDACs). Serotonin (5-HT) is a neuromodulator with neurotransmitter and neuroendocrine functions that contributes to tumorigenesis. We investigated the role of 5-HT signaling in the growth of pancreatic tumors. METHODS: We measured the levels of proteins that regulate 5-HT synthesis, packaging, and degradation in pancreata from KrasG12D/+/Trp53R172H/+/Pdx1-Cre (KPC) mice, which develop pancreatic tumors, as well as in PDAC cell lines and a tissue microarray containing 81 human PDAC samples. We also analyzed expression levels of proteins involved in 5-HT synthesis and degradation by immunohistochemical analysis of a tissue microarray containing 311 PDAC specimens, and associated expression levels with patient survival times. 5-HT level in 14 matched PDAC tumor and non-tumor tissues were analyzed by ELISA. PDAC cell lines were incubated with 5-HT and cell survival and apoptosis were measured. We analyzed expression of the 5-HT receptor HTR2B in PDAC cells and effects of receptor agonists and antagonists, as well as HTR2B knockdown with small hairpin RNAs. We determined the effects of 5-HT stimulation on gene expression profiles of BxPC-3 cells. Regulation of glycolysis by 5-HT signaling via HTR2B was assessed by immunofluorescence and immunoprecipitation analyses, as well as by determination of the extracellular acid ratio, glucose consumption, and lactate production. Primary PDACs, with or without exposure to SB204741 (a selective antagonist of HTR2B), were grown as xenograft tumors in mice, and SB204741 was administered to tumor-bearing KPC mice; tumor growth and metabolism were measured by imaging analyses. RESULTS: In immunohistochemical analysis of a tissue microarray of PDAC specimens, increased levels of TPH1 and decreased level of MAOA, which regulate 5-HT synthesis and degradation, correlated with stage and size of PDACs and shorter patient survival time. We found levels of 5-HT to be increased in human PDAC tissues compared with non-tumor pancreatic tissues, and PDAC cell lines compared with non-transformed pancreatic cells. Incubation of PDAC cell lines with 5-HT increased proliferation and prevented apoptosis. Agonists of HTR2B, but not other 5-HT receptors, promoted proliferation and prevented apoptosis of PDAC cells. Knockdown of HTR2B in PDAC cells, or incubation of cells with HTR2B inhibitors, reduced their growth as xenograft tumors in mice. We observed a correlation between 5-HT and glycolytic flux in PDAC cells; levels of metabolic enzymes involved in glycolysis, the phosphate pentose pathway, and hexosamine biosynthesis pathway increased significantly in PDAC cells following 5-HT stimulation. 5-HT stimulation led to formation of the HTR2B-LYN-p85 complex, which increased PI3K-Akt-mTOR signaling and the Warburg effect by increasing protein levels of MYC and HIF1A. Administration of SB204741 to KPC mice slowed growth and metabolism of established pancreatic tumors and prolonged survival of the mice. CONCLUSIONS: Human PDACs have increased levels of 5-HT, and PDAC cells increase expression of its receptor, HTR2B. These increases allow for tumor glycolysis under metabolic stress and promote growth of pancreatic tumors and PDAC xenograft tumors in mice.