RESUMEN
Malignant tumors, such as colorectal cancer (CRC), are heterogeneous diseases characterized by distinct metabolic phenotypes. These include Warburg- and reverse Warburg phenotypes depending on differential distribution of the lactate carrier proteins monocarboxylate transporter-4 and -1 (MCT4 and MCT1). Here, we elucidated the role of the antioxidant transcription factor nuclear factor E2-related factor-2 (Nrf2) as the key regulator of cellular adaptation to inflammatory/environmental stress in shaping the metabolism toward a reverse Warburg phenotype in malignant and premalignant colonic epithelial cells. Immunohistochemistry of human CRC tissues revealed reciprocal expression of MCT1 and MCT4 in carcinoma and stroma cells, respectively, accompanied by strong epithelial Nrf2 activation. In colorectal tissue from inflammatory bowel disease patients, MCT1 and Nrf2 were coexpressed as well, relating to CD68+inflammatory infiltrates. Indirect coculture of human NCM460 colonocytes with M1- but not M2 macrophages induces MCT1 as well as G6PD, LDHB and TALDO expression, whereas MCT4 expression was decreased. Nrf2 knockdown or reactive oxygen species (ROS) scavenging blocked these coculture effects in NCM460 cells. Likewise, Nrf2 knockdown inhibited similar effects of tBHQ-mediated Nrf2 activation on NCM460 and HCT15 CRC cells. M1 coculture or Nrf2 activation/overexpression greatly altered the lactate uptake but not glucose uptake and mitochondrial activities in these cells, reflecting the reverse Warburg phenotype. Depending on MCT1-mediated lactate uptake, Nrf2 conferred protection from TRAIL-induced apoptosis in NCM460 and HCT15 cells. Moreover, metabolism-dependent clonal growth of HCT15 cells was induced by Nrf2-dependent activation of MCT1-driven lactate exchange. These findings indicate that Nrf2 has an impact on the metabolism already in premalignant colonic epithelial cells exposed to inflammatory M1 macrophages, an effect accompanied by growth and survival alterations. Favoring the reverse Warburg effect, these Nrf2-dependent alterations add to malignant transformation of the colonic epithelium.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/genética , Factor 2 Relacionado con NF-E2/metabolismo , Simportadores/genética , Apoptosis/genética , Biopsia , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Técnicas de Cocultivo , Colon/citología , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibroblastos , Técnicas de Silenciamiento del Gen , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ácido Láctico/metabolismo , Macrófagos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células del Estroma/metabolismo , Células del Estroma/patología , Simportadores/metabolismo , Microambiente Tumoral/genéticaRESUMEN
The altered expression and/or activity of ion channels and transporters (transportome) have been associated with malignant behavior of cancer cells and were proposed to be a hallmark of cancer. However, the impact of altered transportome in epithelial cancers, such as pancreatic ductal adenocarcinoma (PDAC), as well as its pathophysiological consequences, still remains unclear. Here, we report the in silico analysis of 840 transportome genes in PDAC patients' tissues. Our study was focused on the transportome changes and their correlation with functional and behavioral responses in PDAC tumor and stromal compartments. The dysregulated gene expression datasets were filtered using a cut-off of fold-change values ≤-2 or ≥2 (adjusted p value ≤0.05). The dysregulated transportome genes were clearly associated with impaired physiological secretory mechanisms and/or pH regulation, control of cell volume, and cell polarity. Additionally, some down-regulated transportome genes were found to be closely linked to epithelial cell differentiation. Furthermore, the observed decrease in genes coding for calcium and chloride transport might be a mechanism for evasion of apoptosis. In conclusion, the current work provides a comprehensive overview of the altered transportome expression and its association with predicted PDAC malignancy with special focus on the epithelial compartment.
Asunto(s)
Adenocarcinoma/metabolismo , Biología Computacional , Simulación por Computador , Perfilación de la Expresión Génica , Canales Iónicos/genética , Canales Iónicos/metabolismo , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Epitelio/metabolismo , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Microambiente TumoralRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest malignancies with an overall life expectancy of 6 months despite current therapies. NF-κB signalling has been shown to be critical for this profound cell-autonomous resistance against chemotherapeutic drugs and death receptor-induced apoptosis, but little is known about the role of the c-Rel subunit in solid cancer and PDAC apoptosis control. In the present study, by analysis of genome-wide patterns of c-Rel-dependent gene expression, we were able to establish c-Rel as a critical regulator of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in PDAC. TRAIL-resistant cells exhibited a strong TRAIL-inducible NF-κB activity, whereas TRAIL-sensitive cells displayed only a small increase in NF-κB-binding activity. Transfection with siRNA against c-Rel sensitized the TRAIL-resistant cells in a manner comparable to siRNA targeting the p65/RelA subunit. Gel-shift analysis revealed that c-Rel is part of the TRAIL-inducible NF-κB complex in PDAC. Array analysis identified NFATc2 as a c-Rel target gene among the 12 strongest TRAIL-inducible genes in apoptosis-resistant cells. In line, siRNA targeting c-Rel strongly reduced TRAIL-induced NFATc2 activity in TRAIL-resistant PDAC cells. Furthermore, siRNA targeting NFATc2 sensitized these PDAC cells against TRAIL-induced apoptosis. Finally, TRAIL-induced expression of COX-2 was diminished through siRNA targeting c-Rel or NFATc2 and pharmacologic inhibition of COX-2 with celecoxib or siRNA targeting COX-2, enhanced TRAIL apoptosis. In conclusion, we were able to delineate a novel c-Rel-, NFATc2- and COX-2-dependent antiapoptotic signalling pathway in PDAC with broad clinical implications for pharmaceutical intervention strategies.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , FN-kappa B/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-rel/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Antineoplásicos/farmacología , Apoptosis , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/fisiopatología , Línea Celular Tumoral , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Resistencia a Antineoplásicos , Humanos , FN-kappa B/genética , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/fisiopatología , Proteínas Proto-Oncogénicas c-rel/genética , Factor de Transcripción ReIA/metabolismoRESUMEN
Patients with chronic inflammatory bowel disease (IBD) have an increased risk to develop colorectal cancer (CRC) particularly after long duration of the disease. Chronic inflammation of the intestinal mucosa is characterized by a marked enrichment of immune cells such as macrophages as well as by high expression of cytokines and growth factors including transforming growth factor-beta 1 (TGF-ß1). The adhesion molecule L1CAM mediates chemoresistance and migration of tumor cells and is elevated in CRC tissues being associated with metastatic spread and poor prognosis for the patients. In this study, we examine the role of TGF-ß1-induced L1CAM expression and macrophages in malignant transformation of intestinal epithelial cells. We demonstrate that TGF-ß1 stimulation leads to a Slug-dependent upregulation of L1CAM expression already in the colonic intestinal epithelial cell line NCM460 thereby enhancing cell motility and apoptosis resistance. Accordingly, NCM460 cells acquired a migratory and apoptosis-resistant phenotype if transfected with L1CAM. Immunohistochemistry of colonic biopsies revealed considerable L1CAM expression in intestinal epithelial cells in tissues from IBD patients but not in normal colonic tissues. Moreover, L1CAM expression increased with duration of disease being associated with the presence of CD33+ macrophages. Coculture with macrophages generated from monocyte colony-stimulating factor (MCSF)-treated monocytes led to the upregulation of Slug and L1CAM in NCM460 cells thereby elevating cell motility and apoptosis resistance. Pharmacological inhibition of TGF-ß1 signalling abolished expression of Slug and L1CAM in cocultured NCM460 cells resulting in decreased cell migration and apoptosis resistance. In conclusion, these data provide new insights into the mechanisms by which IBD promotes malignant transformation of intestinal epithelial cells and underscore the role of L1CAM and macrophages in this scenario.
Asunto(s)
Apoptosis , Transformación Celular Neoplásica , Mucosa Intestinal/metabolismo , Macrófagos/fisiología , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Adulto , Anciano , Línea Celular , Movimiento Celular , Transformación Celular Neoplásica/genética , Técnicas de Cocultivo , Neoplasias Colorrectales/patología , Células Epiteliales/metabolismo , Femenino , Humanos , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/genética , Interferencia de ARN , ARN Interferente Pequeño , Lectina 3 Similar a Ig de Unión al Ácido Siálico/biosíntesis , Transducción de Señal , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/genética , Adulto JovenRESUMEN
Evidence accumulates that the transcription factor nuclear factor E2-related factor 2 (Nrf2) has an essential role in cancer development and chemoresistance, thus pointing to its potential as an anticancer target and undermining its suitability in chemoprevention. Through the induction of cytoprotective and proteasomal genes, Nrf2 confers apoptosis protection in tumor cells, and inhibiting Nrf2 would therefore be an efficient strategy in anticancer therapy. In the present study, pancreatic carcinoma cell lines (Panc1, Colo357 and MiaPaca2) and H6c7 pancreatic duct cells were analyzed for the Nrf2-inhibitory effect of the coffee alkaloid trigonelline (trig), as well as for its impact on Nrf2-dependent proteasome activity and resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and anticancer drug-induced apoptosis. Chemoresistant Panc1 and Colo357 cells exhibit high constitutive Nrf2 activity, whereas chemosensitive MiaPaca2 and H6c7 cells display little basal but strong tert-butylhydroquinone (tBHQ)-inducible Nrf2 activity and drug resistance. Trig efficiently decreased basal and tBHQ-induced Nrf2 activity in all cell lines, an effect relying on a reduced nuclear accumulation of the Nrf2 protein. Along with Nrf2 inhibition, trig blocked the Nrf2-dependent expression of proteasomal genes (for example, s5a/psmd4 and α5/psma5) and reduced proteasome activity in all cell lines tested. These blocking effects were absent after treatment with Nrf2 siRNA, a condition in which proteasomal gene expression and proteasome activity were already decreased, whereas siRNA against the related transcription factor Nrf1 did not affect proteasome activity and the inhibitory effect of trig. Depending on both Nrf2 and proteasomal gene expression, the sensitivity of all cell lines to anticancer drugs and TRAIL-induced apoptosis was enhanced by trig. Moreover, greater antitumor responses toward anticancer drug treatment were observed in tumor-bearing mice when receiving trig. In conclusion, representing an efficient Nrf2 inhibitor capable of blocking Nrf2-dependent proteasome activity and thereby apoptosis protection in pancreatic cancer cells, trig might be beneficial in improving anticancer therapy.
Asunto(s)
Alcaloides/farmacología , Apoptosis , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Neoplasias Pancreáticas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Línea Celular Tumoral , Etopósido/farmacología , Humanos , Ratones , Ratones SCID , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Complejo de la Endopetidasa Proteasomal/genética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The constitutive activation of the transcription factor nuclear-factor kappa B (NF-kappaB) is a hallmark of many highly malignant tumours such as the pancreatic ductal adenocarcinoma and accounts for profound chemoresistance. Inhibition of NF-kappaB activation has been shown to be a useful strategy for increasing the sensitivity towards cytostatic drug treatment in vitro and in vivo. Moreover, various pharmacological substances (e.g. thalidomide, bortezomib, sulphasalazine) have already entered clinical studies partially showing promising results for certain types of cancer. Further studies will be needed, in particular for pancreatic ductal adenocarcinoma, to evaluate the therapeutic efficacy of appropriate combinations of a NF-kappaB inhibitor and cytostatic drugs.