RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers in western countries, with a median survival of 6 months and an extremely low percentage of long-term surviving patients. KRAS mutations are known to be a driver event of PDAC, but targeting mutant KRAS has proved challenging. Targeting oncogene-driven signalling pathways is a clinically validated approach for several devastating diseases. Still, despite marked tumour shrinkage, the frequency of relapse indicates that a fraction of tumour cells survives shut down of oncogenic signalling. Here we explore the role of mutant KRAS in PDAC maintenance using a recently developed inducible mouse model of mutated Kras (Kras(G12D), herein KRas) in a p53(LoxP/WT) background. We demonstrate that a subpopulation of dormant tumour cells surviving oncogene ablation (surviving cells) and responsible for tumour relapse has features of cancer stem cells and relies on oxidative phosphorylation for survival. Transcriptomic and metabolic analyses of surviving cells reveal prominent expression of genes governing mitochondrial function, autophagy and lysosome activity, as well as a strong reliance on mitochondrial respiration and a decreased dependence on glycolysis for cellular energetics. Accordingly, surviving cells show high sensitivity to oxidative phosphorylation inhibitors, which can inhibit tumour recurrence. Our integrated analyses illuminate a therapeutic strategy of combined targeting of the KRAS pathway and mitochondrial respiration to manage pancreatic cancer.
Asunto(s)
Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Mitocondrias/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Autofagia , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Respiración de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Genes p53/genética , Glucólisis , Lisosomas/metabolismo , Ratones , Mitocondrias/efectos de los fármacos , Mutación/genética , Recurrencia Local de Neoplasia/prevención & control , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosforilación Oxidativa/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Recurrencia , Transducción de Señal , Neoplasias PancreáticasRESUMEN
Cancer cells have metabolic dependencies that distinguish them from their normal counterparts. Among these dependencies is an increased use of the amino acid glutamine to fuel anabolic processes. Indeed, the spectrum of glutamine-dependent tumours and the mechanisms whereby glutamine supports cancer metabolism remain areas of active investigation. Here we report the identification of a non-canonical pathway of glutamine use in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for tumour growth. Whereas most cells use glutamate dehydrogenase (GLUD1) to convert glutamine-derived glutamate into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid cycle, PDAC relies on a distinct pathway in which glutamine-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate by aspartate transaminase (GOT1). Subsequently, this oxaloacetate is converted into malate and then pyruvate, ostensibly increasing the NADPH/NADP(+) ratio which can potentially maintain the cellular redox state. Importantly, PDAC cells are strongly dependent on this series of reactions, as glutamine deprivation or genetic inhibition of any enzyme in this pathway leads to an increase in reactive oxygen species and a reduction in reduced glutathione. Moreover, knockdown of any component enzyme in this series of reactions also results in a pronounced suppression of PDAC growth in vitro and in vivo. Furthermore, we establish that the reprogramming of glutamine metabolism is mediated by oncogenic KRAS, the signature genetic alteration in PDAC, through the transcriptional upregulation and repression of key metabolic enzymes in this pathway. The essentiality of this pathway in PDAC and the fact that it is dispensable in normal cells may provide novel therapeutic approaches to treat these refractory tumours.
Asunto(s)
Glutamina/metabolismo , Redes y Vías Metabólicas , Proteína Oncogénica p21(ras)/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas ras/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Aspartato Aminotransferasas/deficiencia , Aspartato Aminotransferasas/genética , Aspartato Aminotransferasas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Ciclo del Ácido Cítrico , Glutamato Deshidrogenasa/metabolismo , Homeostasis , Humanos , Ácidos Cetoglutáricos/metabolismo , Proteína Oncogénica p21(ras)/genética , Oncogenes/genética , Oxidación-Reducción , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Especies Reactivas de Oxígeno/metabolismo , Proteínas ras/genéticaRESUMEN
BACKGROUND: Wisteria floribunda agglutinin-positive mac-2 binding protein (WFA+-M2BP) is an excellent biomarker for predicting hepatic fibrosis. We hypothesized WFA+-M2BP might be a serum biomarker for the diagnosis of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC) with dense fibrosis. METHODS: In this study, we included 16 CP and 24 PDAC patients. Serum levels of WFA+-M2BP (cut-off index [COI]) were compared between the 2 groups. To confirm the cellular production of WFA+-M2BP, we investigated the presence of WFA+-M2BP in HEK293 cells, 3 established human PDAC cell lines and a recently generated human PDAC cell line derived from a liver metastasis (MDA-PATC53). The bio-physiological effects of MDA-PATC53 supernatant were evaluated. Finally, the difference in the expression of glycosylation enzymes between MDA-PATC53 and Panc-1 were analyzed by cDNA microarray. RESULTS: We found that the serum WFA+-M2BP level could distinguish the 2 groups. The median serum COI of WFA+-M2BP was 0.98 and 0.51 in PDAC and CP, respectively. Additionally, WFA+-M2BP positive PDACs were more frequently associated with metastatic lesions than the WFA+-M2BP negative PDACs (91.6% vs. 41.7%, P = 0.009). The MDA-PATC53 cells alone produced WFA+-M2BP. However, we found that MDA-PATC53 supernatant containing WFA+-M2BP (1.0 COI) did not alter the biological behavior of cancer cell lines. The results of cDNA microarray revealed that several glycosylation enzymes with pro-oncologic function were highly expressed in MDA-PATC53 compared to Panc-1. CONCLUSIONS: Serum WFA+-M2BP can be a useful biomarker for the diagnosis of PDAC and the prediction of disease progression since it potentially reflects altered pro-oncologic glycosylation enzymes.
Asunto(s)
Antígenos de Neoplasias/sangre , Carcinoma Ductal Pancreático/sangre , Glicoproteínas de Membrana/sangre , Neoplasias Pancreáticas/sangre , Lectinas de Plantas , Receptores N-Acetilglucosamina , Biomarcadores de Tumor/sangre , Carcinoma Ductal Pancreático/patología , Movimiento Celular , ADN de Neoplasias/genética , Fibrosis , Células HEK293 , Humanos , Análisis por Micromatrices , Invasividad Neoplásica/patología , Neoplasias Pancreáticas/patología , Pancreatitis Crónica/sangre , Pancreatitis Crónica/diagnósticoRESUMEN
Many commercially available cell lines have been in culture for ages, acquiring phenotypes that differ from the original cancers from which these cell lines were derived. Therefore, research on new cell lines could improve the success rates of translational research in cancer. We have developed methods for the isolation and culture of human pancreatic ductal adenocarcinoma (PDAC) cells from murine xenografts of human PDAC. We hypothesize that phenotypes of PDAC cells are modified by in vitro culture conditions over time and by in vivo implantation. Patient-derived xenografts were created in immunodeficient mice using surgically resected tumor specimens. These murine xenografts were then used to establish human PDAC cell lines in culture. Earlier (<5) passage and later (>20) passage cell lines were evaluated separately regarding proliferation, cell cycle, genetic mutations, invasiveness, chemosensitivity, tumorigenesis, epithelial-mesenchymal transition (EMT) status, and proteomics. Later passage cells accelerated their doubling time and colony formation, and were more concentrated in the G0/G1 phase and less in the G2/M checkpoint phase. Later passage cells were more sensitive to gemcitabine and 5-fluorouracil than earlier passage cells, but all four new cell lines were more chemo-resistant compared with commercial ATCC cell lines. EMT induction was observed when establishing and passaging cell lines in vitro and furthermore by growing them as subcutaneous tumors in vivo. This study demonstrates a novel approach to the establishment of PDAC cell lines and observes a process by which newly established cell lines undergo phenotypic changes during in vitro culture and in vivo tumorigenesis. This may help explain differences of treatment effects often observed between experiments conducted in vitro, in vivo, and in human clinical trials.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Transición Epitelial-Mesenquimal/fisiología , Neoplasias Pancreáticas/fisiopatología , Fenotipo , Animales , Western Blotting , Ciclo Celular/fisiología , Proliferación Celular/fisiología , Ensayo de Unidades Formadoras de Colonias , Desoxicitidina/análogos & derivados , Fluorouracilo , Xenoinjertos/citología , Xenoinjertos/fisiología , Humanos , Inmunohistoquímica , Ratones , Invasividad Neoplásica/fisiopatología , Análisis por Matrices de Proteínas , Proteómica/métodos , Células Tumorales Cultivadas , Gemcitabina , Neoplasias PancreáticasRESUMEN
BACKGROUND: Recurrence after resection of pancreatic ductal adenocarcinoma (PDAC) is common, thus postoperative surveillance is critical for detection and treatment of recurrent disease. The development of biologically based techniques for early recurrence detection may enable more timely and effective treatment of such recurrences. METHODS: Tumor fragments derived from patients who underwent potentially curative resection of PDAC were heterotopically implanted into NOD/SCID mice. Engraftment success rates and growth parameters were matched to clinicopathologic data, preoperative treatment status, and oncologic outcomes to correlate disease-free survival (DFS) and overall survival. RESULTS: Seventy patients consented to participate with 56 (80 %) developing a mouse PDAC tumorgraft. Patients with successful engraftment had a shorter median DFS compared with patients whose tumorgrafts failed to engraft (9.8 vs. 40.9 mo, respectively; p < 0.01). Fifty patients received preoperative therapy with 36 (72 %) successful tumorgrafts from this cohort. On multivariate analysis, lymph node metastasis (hazard ratio [HR] 3, 95 % CI 1.4-6.7, p < 0.01) and successful engraftment (HR 5.8, 95 % CI 2-16.9, p < 0.01) were predictive of a shorter DFS in the preoperative therapy cohort. In patients who recurred, tumorgraft formation was identified at a median of 134.5 days before standard methods of radiographic recurrence detection (p < 0.01). CONCLUSIONS: Patient-derived tumorgrafts from resected PDAC may potentially predict recurrence months before currently available surveillance modalities. This lead-time advantage may allow for earlier implementation or changes in therapy as successful engraftment, particularly in those having undergone preoperative therapy, may indicate a more biologically aggressive disease.
Asunto(s)
Carcinoma Ductal Pancreático/patología , Recurrencia Local de Neoplasia/patología , Pancreatectomía/mortalidad , Neoplasias Pancreáticas/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/cirugía , Femenino , Estudios de Seguimiento , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Invasividad Neoplásica , Recurrencia Local de Neoplasia/mortalidad , Recurrencia Local de Neoplasia/cirugía , Estadificación de Neoplasias , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/cirugía , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
There is substantial heterogeneity in the clinical behavior of pancreatic cancer and in its response to therapy. Some of this variation may be due to differences in delivery of cytotoxic therapies between patients and within individual tumors. Indeed, in 12 patients with resectable pancreatic cancer, we previously demonstrated wide inter-patient variability in the delivery of gemcitabine as well as in the mass transport properties of tumors as measured by computed tomography (CT) scans. However, the variability of drug delivery and transport properties within pancreatic tumors is currently unknown. Here, we analyzed regional measurements of gemcitabine DNA incorporation in the tumors of the same 12 patients to understand the degree of intra-tumoral heterogeneity of drug delivery. We also developed a volumetric segmentation approach to measure mass transport properties from the CT scans of these patients and tested inter-observer agreement with this new methodology. Our results demonstrate significant heterogeneity of gemcitabine delivery within individual pancreatic tumors and across the patient cohort, with gemcitabine DNA incorporation in the inner portion of the tumors ranging from 38 to 74% of the total. Similarly, the CT-derived mass transport properties of the tumors had a high degree of heterogeneity, ranging from minimal difference to almost 200% difference between inner and outer portions of the tumor. Our quantitative method to derive transport properties from CT scans demonstrated less than 5% difference in gemcitabine prediction at the average CT-derived transport value across observers. These data illustrate significant inter-patient and intra-tumoral heterogeneity in the delivery of gemcitabine, and highlight how this variability can be reproducibly accounted for using principles of mass transport. With further validation as a biophysical marker, transport properties of tumors may be useful in patient selection for therapy and prediction of therapeutic outcome.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacocinética , Desoxicitidina/análogos & derivados , Sistemas de Liberación de Medicamentos , Neoplasias Pancreáticas/metabolismo , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/metabolismo , Transporte Biológico , Aductos de ADN/metabolismo , ADN de Neoplasias/metabolismo , Desoxicitidina/administración & dosificación , Desoxicitidina/metabolismo , Desoxicitidina/farmacocinética , Humanos , Inyecciones Intravenosas , Pancreatectomía , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Distribución Tisular , Tomógrafos Computarizados por Rayos X , Microambiente Tumoral , GemcitabinaRESUMEN
[This retracts the article DOI: 10.7150/ijbs.22555.].
RESUMEN
Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Ratones , Animales , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismoRESUMEN
Feedback regulation of transcription factor NF-kappaB by its inhibitor IkappaBalpha plays an essential role in control of NF-kappaB activity. To understand the biological significance of IkappaBalpha-mediated feedback regulation of NF-kappaB, we generated mice harboring mutated kappaB enhancers in the promoter of the IkappaBalpha gene (IkappaBalpha(M/M)) to inhibit NF-kappaB-regulated IkappaBalpha expression. Here, we report that these mutant mice are defective in NF-kappaB-induced expression of IkappaBalpha. This defective feedback regulation of NF-kappaB by IkappaBalpha not only altered activity of NF-kappaB, but also the expression of NF-kappaB-regulated genes. As a result, IkappaBalpha(M/M), the homozygous knock-in mice with mutated kappaB enhancers in the IkappaBalpha promoter, acquire shorten life span, hypersensitivity to septic shock, abnormal T-cell development and activation, and Sjögren's Syndrome. These findings therefore demonstrate that the IkappaBalpha-mediated feedback regulation of NF-kappaB has an essential role in controlling T-cell development and functions, provide mechanistic insight into the development of Sjögren's Syndrome, and suggest the potential of NF-kappaB signaling as a therapeutic target for Sjögren's Syndrome and other autoimmune diseases.
Asunto(s)
Proteínas I-kappa B/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Síndrome de Sjögren/genética , Síndrome de Sjögren/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos , Retroalimentación Fisiológica , Expresión Génica , Técnicas de Sustitución del Gen , Humanos , Técnicas In Vitro , Activación de Linfocitos , Ratones , Ratones Mutantes , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Inhibidor NF-kappaB alfa , Regiones Promotoras Genéticas , Transducción de Señal , Síndrome de Sjögren/inmunología , Síndrome de Sjögren/patología , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
BACKGROUND: Pancreatic adenocarcinoma is among the most resistant of human cancers, yet specific mechanisms of treatment resistance remain poorly understood. Models to study pancreatic cancer resistance remain limited and should reflect in vivo changes that occur within patient tumors. We sought to identify consistent, differentially expressed genes between treatment of naive pancreatic tumors and those exposed to neoadjuvant therapy using a strict, in vivo direct xenograft model system. METHODS: Over a 42-week period, 12 untreated and treated patient tumors were successfully engrafted into NOD/SCID mice. RNA from each treatment group (5 untreated and 4 treated) was isolated in triplicate and subjected to global gene expression analysis. Consistent gene expression changes with treatment were identified and confirmed using RT-PCR and immunohistochemistry. RESULTS: Engraftment of untreated patient tumors was more frequent than treated tumors (17 of 21 versus 16 of 49, P = .0002) but without differences in observed time until tumor formation. The histology of patient tumors was recapitulated in direct xenograft tumors. Relative to untreated tumors, treated tumors consistently demonstrated more than a 2-fold reduction in TGFß-R2 mRNA expression and more than a 5-fold increase in IGFBP3 expression (P < .0218) and were confirmed by immunohistochemistry. CONCLUSION: Engraftment of human pancreatic tumors into immunodeficient mice prior to and following neoadjuvant therapy is possible and provides an in vivo platform for comparison of global gene expression patterns. The decreased TGFß-R2 expression and increased IGFBP3 expression among direct xenograft tumors derived from treated tumors relative to untreated tumors suggests a role in therapy resistance and warrants further study.
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Adenocarcinoma/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Neoplasias Pancreáticas/genética , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Animales , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Terapia Neoadyuvante , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Trasplante HeterólogoRESUMEN
[This retracts the article DOI: 10.3892/ol.2018.8219.].
RESUMEN
OBJECTIVE: Intra-tumoral expression of the serine hydrolase carboxylesterase 2 (CES2) contributes to the activation of the pro-drug irinotecan in pancreatic ductal adenocarcinoma (PDAC). Given other potential roles of CES2, we assessed its regulation, downstream effects, and contribution to tumor development in PDAC. METHODS: Association between the mRNA expression of CES2 in pancreatic tumors and overall survival was assessed using The Cancer Genome Atlas. Cell viability, clonogenic, and anchorage-independent growth assays as well as an orthotopic mouse model of PDAC were used to evaluate the biological relevance of CES2 in pancreatic cancer. CES2-driven metabolic changes were determined by untargeted and targeted metabolomic analyses. RESULTS: Elevated tumoral CES2 mRNA expression was a statistically significant predictor of poor overall survival in PDAC patients. Knockdown of CES2 in PDAC cells reduced cell viability, clonogenic capacity, and anchorage-independent growth in vitro and attenuated tumor growth in an orthotopic mouse model of PDAC. Mechanistically, CES2 was found to promote the catabolism of phospholipids resulting in HNF4α activation through a soluble epoxide hydrolase (sEH)-dependent pathway. Targeting of CES2 via siRNA or small molecule inhibitors attenuated HNF4α protein expression and reduced gene expression of classical/progenitor markers and increased basal-like markers. Targeting of the CES2-sEH-HNF4α axis using small molecule inhibitors of CES2 or sEH reduced cell viability. CONCLUSIONS: We establish a novel regulatory loop between CES2 and HNF4α to sustain the progenitor subtype and promote PDAC progression and highlight the potential utility of CES2 or sEH inhibitors for the treatment of PDAC as part of non-irinotecan-containing regimens.
Asunto(s)
Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Adenocarcinoma/genética , Animales , Carboxilesterasa/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/uso terapéutico , Humanos , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismoRESUMEN
BACKGROUNDPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with unpredictable responses to chemotherapy. Approaches to assay patient tumors before treatment and identify effective treatment regimens based on tumor sensitivities are lacking. We developed an organoid-based platform (OBP) to visually quantify patient-derived organoid (PDO) responses to drug treatments and associated tumor-stroma modulation for personalized PDAC therapy.METHODSWe retrospectively quantified apoptotic responses and tumor-stroma cell proportions in PDOs via 3D immunofluorescence imaging through annexin A5, α-smooth muscle actin (α-SMA), and cytokeratin 19 (CK-19) levels. Simultaneously, an ex vivo organoid drug sensitivity assay (ODSA) was used to measure responses to standard-of-care regimens. Differences between ODSA results and patient tumor responses were assessed by exact McNemar's test.RESULTSImmunofluorescence signals, organoid growth curves, and Ki-67 levels were measured and authenticated through the OBP for up to 14 days. ODSA drug responses were not different from patient tumor responses, as reflected by CA19-9 reductions following neoadjuvant chemotherapy (P = 0.99). PDOs demonstrated unique apoptotic and tumor-stroma modulation profiles (P < 0.0001). α-SMA/CK-19 ratio levels of more than 1.0 were associated with improved outcomes (P = 0.0179) and longer parental patient survival by Kaplan-Meier analysis (P = 0.0046).CONCLUSIONHeterogenous apoptotic drug responses and tumor-stroma modulation are present in PDOs after standard-of-care chemotherapy. Ratios of α-SMA and CK-19 levels in PDOs are associated with patient survival, and the OBP could aid in the selection of personalized therapies to improve the efficacy of systemic therapy in patients with PDAC.FUNDINGNIH/National Cancer Institute grants (K08CA218690, P01 CA117969, R50 CA243707-01A1, U54CA224065), the Skip Viragh Foundation, the Bettie Willerson Driver Cancer Research Fund, and a Cancer Center Support Grant for the Flow Cytometry and Cellular Imaging Core Facility (P30CA16672).
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Medicina de Precisión , Estudios Retrospectivos , Imagenología Tridimensional , Carcinoma Ductal Pancreático/diagnóstico por imagen , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Organoides/patología , Neoplasias PancreáticasRESUMEN
Tumor-associated macrophages (TAMs) represent the M2-like phenotype with potent immunosuppressive activity, and play a pro-tumor role in pancreatic ductal adenocarcinoma (PDAC) biology. In this study, we investigated the role of the insulin-like growth factor binding protein 2 (IGFBP2) as a determinant of TAM polarity. Clinical data revealed that the levels of IGFBP2 correlated with M2 TAMs accumulation and disease progression in human PDAC. In vivo mouse model experiments showed that IGFBP2 promoted an immunosuppressive microenvironment and tumor growth in a macrophage dependent manner. Bioinformatics analysis of PDAC transcriptomes revealed a significant association between IGFBP2 expression and M2 macrophage polarization and signal transducer and activator of transcription 3 (STAT3) activation. Mechanistic investigations demonstrated that IGFBP2 augmented the expression and secretion of IL-10 through STAT3 activation in PDAC cells, which induced TAM polarization toward an M2 phenotype. IGFBP2-polarized M2 macrophages significantly increased Tregs infiltration and impaired antitumor T-cell immunity in a mouse model. Thus, our investigations have illuminated the IGFBP2 signaling pathway that contributes to the macrophage-based immunosuppressive microenvironment in PDAC, suggesting that blocking the IGFBP2 axis constitutes a potential treatment strategy to reset TAM polarization toward an antitumor state in PDAC.
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Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor de Transcripción STAT3/genética , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Polaridad Celular/genética , Proliferación Celular/genética , Progresión de la Enfermedad , Xenoinjertos , Humanos , Ratones , Transducción de Señal/genética , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/patologíaRESUMEN
Nuclear factor erythroid 2-related factor 2 (NRF2) is aberrantly activated in about 93% of pancreatic cancers. Activated NRF2 regulates multiple downstream molecules involved in cancer cell metabolic reprogramming, translational control, and treatment resistance; however, targeting NRF2 for pancreatic cancer therapy remains largely unexplored. In this study, we used the online computational tool CellMinerTM to explore the NCI-60 drug databases for compounds with anticancer activities correlating most closely with the mRNA expression of NQO1, a marker for NRF2 pathway activity. Among the >100,000 compounds analyzed, NSC84167, termed herein as NRF2 synthetic lethality compound-01 (NSLC01), was one of the top hits (r = 0.71, P < 0.001) and selected for functional characterization. NSLC01 selectively inhibited the viabilities of four out of seven conventional pancreatic cancer cell lines and induced dramatic apoptosis in the cells with high NRF2 activation. The selective anticancer activity of NSLC01 was further validated with a panel of nine low-passage pancreatic patient-derived cell lines, and a significant reverse correlation between log(IC50) of NSLC01 and NQO1 expression was confirmed (r = -0.5563, P = 0.024). Notably, screening of a panel of nine patient-derived xenografts (PDXs) revealed six PDXs with high NQO1/NRF2 activation, and NSLC01 dramatically inhibited the viabilities and induced apoptosis in ex vivo cultures of PDX tumors. Consistent with the ex vivo results, NSLC01 inhibited the tumor growth of two NRF2-activated PDX models in vivo (P < 0.01, n = 7-8) but had no effects on the NRF2-low counterpart. To characterize the mechanism of action, we employed a metabolomic isotope tracer assay that demonstrated that NSLC01-mediated inhibition of de novo synthesis of multiple amino acids, including asparagine and methionine. Importantly, we further found that NSLC01 suppresses the eEF2K/eEF2 translation elongation cascade and protein translation of asparagine synthetase. In summary, this study identified a novel compound that selectively targets protein translation and induces synthetic lethal effects in NRF2-activated pancreatic cancers.
Asunto(s)
Antineoplásicos/farmacología , Asparagina/biosíntesis , Aspartatoamoníaco Ligasa/metabolismo , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa del Factor 2 de Elongación/metabolismo , Humanos , Ratones Endogámicos NOD , Ratones SCID , NAD(P)H Deshidrogenasa (Quinona)/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. A critical unmet need exists to explore essential susceptibilities in PDAC and to identify druggable targets to improve PDAC treatment. KRAS mutations dominate the genetic landscape of PDAC and lead to activation of multiple downstream pathways and cellular processes. Here, we investigated the requirement of these pathways for tumor maintenance using an inducible KrasG12D -driven PDAC mouse model (iKras model), identifying that RAF-MEK-MAPK signaling is the major effector for oncogenic KRAS-mediated tumor maintenance. However, consistent with previous studies, MEK inhibition had minimal therapeutic effect as a single agent for PDAC in vitro and in vivo. Although MEK inhibition partially downregulated transcription of glycolysis genes, it failed to suppress glycolytic flux in PDAC cells, which is a major metabolic effector of oncogenic KRAS. Accordingly, an in vivo genetic screen identified multiple glycolysis genes as potential targets that may sensitize tumor cells to MEK inhibition. Inhibition of glucose metabolism with low-dose 2-deoxyglucose in combination with a MEK inhibitor induced apoptosis in KrasG12D -driven PDAC cells in vitro. The combination also inhibited xenograft PDAC tumor growth and prolonged overall survival in a genetically engineered PDAC mouse model. Molecular and metabolic analyses indicated that co-targeting glycolysis and MAPK signaling results in apoptosis via induction of lethal endoplasmic reticulum stress. Together, our work suggests that combined inhibition of glycolysis and the MAPK pathway may serve as an effective approach to target KRAS-driven PDAC. SIGNIFICANCE: This study demonstrates the critical role of glucose metabolism in resistance to MAPK inhibition in KRAS-driven pancreatic cancer, uncovering a potential therapeutic approach for treating this aggressive disease.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Carcinoma Ductal Pancreático/tratamiento farmacológico , Glucosa/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Humanos , Ratones , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most TP53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis, yet the extent of direct cooperation between KRAS effectors and mutant p53 remains largely undefined. We show that oncogenic KRAS effectors activate CREB1 to allow physical interactions with mutant p53 that hyperactivate multiple prometastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the prometastatic, pioneer transcription factor FOXA1, activating its transcriptional network while promoting WNT/ß-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced FOXA1 and ß-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic KRAS and mutant p53 to mitigate metastasis. SIGNIFICANCE: Oncogenic KRAS and mutant p53 are the most commonly mutated oncogene and tumor suppressor gene in human cancers, yet direct interactions between these genetic drivers remain undefined. We identified a cooperative node between oncogenic KRAS effectors and mutant p53 that can be therapeutically targeted to undermine cooperation and mitigate metastasis.This article is highlighted in the In This Issue feature, p. 1861.
Asunto(s)
Carcinoma Ductal Pancreático/genética , Genes p53/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Carcinoma Ductal Pancreático/patología , Femenino , Redes Reguladoras de Genes , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Metástasis de la Neoplasia , Neoplasias Pancreáticas/patologíaRESUMEN
Despite efforts in understanding its underlying mechanisms, the etiology of chromosomal instability (CIN) remains unclear for many tumor types. Here, we identify CIN initiation as a previously undescribed function for APOBEC3A (A3A), a cytidine deaminase upregulated across cancer types. Using genetic mouse models of pancreatic ductal adenocarcinoma (PDA) and genomics analyses in human tumor cells we show that A3A-induced CIN leads to aggressive tumors characterized by enhanced early dissemination and metastasis in a STING-dependent manner and independently of the canonical deaminase functions of A3A. We show that A3A upregulation recapitulates numerous copy number alterations commonly observed in patients with PDA, including co-deletions in DNA repair pathway genes, which in turn render these tumors susceptible to poly (ADP-ribose) polymerase inhibition. Overall, our results demonstrate that A3A plays an unexpected role in PDA as a specific driver of CIN, with significant effects on disease progression and treatment.
Asunto(s)
Citidina Desaminasa , Neoplasias Pancreáticas , Animales , Inestabilidad Cromosómica/genética , Citidina Desaminasa/genética , Humanos , Ratones , Neoplasias Pancreáticas/genética , Proteínas/genética , Neoplasias PancreáticasRESUMEN
The aim of the study was to investigate the effects of lactic acid on the phenotypic polarization and immune function of macrophages. The human monocyte/macrophage cell line, THP1, was selected and treated with lactic acid. Immunofluorescence staining, laser confocal microscopy, reversetranscription polymerase chain reaction (RTPCR), western blot, siRNA, and ELISA analyses were used to observe changes in the levels of cluster of differentiation (CD)68, CD163, hypoxia inducible factor (HIF)1α, and programmed death ligand1 (PDL1) as well as those of cytokines, tumor necrosis factor (TNF)α, interferon (IFN)γ, interleukin (IL)12, and IL10. THP1 macrophages and T cells were cocultured in vitro to observe the changes in proliferation and apoptosis of T cells. The results showed that, lactic acid (15 mmol/l) significantly upregulated the expression of the macrophage M2 marker CD163 (P<0.05), cytokines, IFNγ and IL10, secreted by M2tumorassociated macrophages (TAM, P<0.05), and HIF1α and PDL1 (P<0.05), and downregulated the expression of cytokines, TNFα and IL12, secreted by M1TAM (P<0.05). Redistribution of M2TAM subsets and PDL1 expression was reversed after further transfection of THP1 cells with HIF1α siRNA (P<0.05). After coculturing, Tcell proliferation was inhibited and apoptosis was promoted. In summary, modulation of lactic acid level can redistribute M2TAM subsets and upregulate PDL1 to assist tumor immune escape. The HIF1α signaling pathway may participate in this process, revealing that macrophages, as 'checkpoints' in organisms, are links that connect the immune status and tumor evolution, and can be used as a target in tumor treatment.
Asunto(s)
Ácido Láctico/metabolismo , Neoplasias/inmunología , Transducción de Señal/inmunología , Linfocitos T/patología , Macrófagos Asociados a Tumores/inmunología , Apoptosis/inmunología , Antígeno B7-H1/metabolismo , Técnicas de Cultivo de Célula , Proliferación Celular , Técnicas de Cocultivo , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/patología , Receptor de Muerte Celular Programada 1/metabolismo , Transducción de Señal/genética , Linfocitos T/inmunología , Células THP-1 , Escape del Tumor , Microambiente Tumoral/inmunología , Macrófagos Asociados a Tumores/metabolismo , Regulación hacia ArribaRESUMEN
PURPOSE: The combination chemotherapy of fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) has provided clinically meaningful improvement for pancreatic ductal adenocarcinoma (PDAC). We previously uncovered a role for the serine hydrolase carboxylesterase 2 (CES2) in mediating intratumoral activation of the prodrug irinotecan, a constituent of FOLFIRINOX. We aimed to further test the predictive value of CES2 for response to irinotecan using patient-derived xenograft (PDX) models and to elucidate the determinants of CES2 expression and response to FOLFIRINOX treatment among patients with PDAC. METHODS: PDXs were engrafted subcutaneously into nude mice and treated for 4 weeks with either saline control or irinotecan. CES2 and hepatocyte nuclear factor 4 alpha (HNF4A) expression in PDAC tissues was evaluated by immunohistochemical and Western blot analysis. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and hemoglobin A1C (HbA1C) levels in patients who underwent neoadjuvant FOLFIRINOX treatment. RESULTS: High CES2 activity in PDAC PDXs was associated with increased sensitivity to irinotecan. Integrated gene expression, proteomic analyses, and in vitro genetic experiments revealed that nuclear receptor HNF4A, which is upregulated in diabetes, is the upstream transcriptional regulator of CES2 expression. Elevated CES2 protein expression in PDAC tissues was positively associated with a history of type 2 diabetes (odds ratio, 4.84; P = .02). High HbA1C levels were associated with longer overall survival in patients who received neoadjuvant FOLFIRINOX treatment (P = .04). CONCLUSION: To our knowledge, we provide, for the first time, evidence that CES2 expression is associated with a history of type 2 diabetes in PDAC and that elevated HbA1C, by predicting tumor CES2 expression, may represent a novel marker for stratifying patients most likely to respond to FOLFIRINOX therapy.