RESUMEN
BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplastic stroma surrounding most tumors. Activated stromal fibroblasts, namely cancer-associated fibroblasts (CAFs), play a major role in PDAC progression. We analyzed whether CAFs influence acinar cells and impact PDAC initiation, that is, acinar-to-ductal metaplasia (ADM). ADM connection with PDAC pathophysiology is indicated, but not yet established. We hypothesized that CAF secretome might play a significant role in ADM in PDAC initiation. METHODS: Mouse and human acinar cell organoids, acinar cells cocultured with CAFs and exposed to CAF-conditioned media, acinar cell explants, and CAF cocultures were examined by means of quantitative reverse transcription polymerase chain reaction, RNA sequencing, immunoblotting, and confocal microscopy. Data from liquid chromatography with tandem mass spectrometry analysis of CAF-conditioned medium and RNA sequencing data of acinar cells post-conditioned medium exposure were integrated using bioinformatics tools to identify the molecular mechanism for CAF-induced ADM. Using confocal microscopy, immunoblotting, and quantitative reverse transcription polymerase chain reaction analysis, we validated the depletion of a key signaling axis in the cell line, acinar explant coculture, and mouse cancer-associated fibroblasts (mCAFs). RESULTS: A close association of acino-ductal markers (Ulex europaeus agglutinin 1, amylase, cytokeratin-19) and mCAFs (α-smooth muscle actin) in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1Cre (KPC) and LSL-KrasG12D/+; Pdx1Cre (KC) autochthonous progression tumor tissue was observed. Caerulein treatment-induced mCAFs increased cytokeratin-19 and decreased amylase in wild-type and KC pancreas. Likewise, acinar-mCAF cocultures revealed the induction of ductal transdifferentiation in cell line, acinar-organoid, and explant coculture formats in WT and KC mice pancreas. Proteomic and transcriptomic data integration revealed a novel laminin α5/integrinα4/stat3 axis responsible for CAF-mediated acinar-to-ductal cell transdifferentiation. CONCLUSIONS: Results collectively suggest the first evidence for CAF-influenced acino-ductal phenotypic switchover, thus highlighting the tumor microenvironment role in pancreatic carcinogenesis inception.
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Células Acinares , Fibroblastos Asociados al Cáncer , Carcinoma Ductal Pancreático , Transdiferenciación Celular , Laminina , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Células Acinares/metabolismo , Células Acinares/patología , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Técnicas de Cocultivo , Medios de Cultivo Condicionados/metabolismo , Metaplasia/patología , Metaplasia/metabolismo , Organoides/metabolismo , Organoides/patología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Transducción de Señal , Microambiente TumoralRESUMEN
BACKGROUND & AIMS: Secreted mucin 5AC (MUC5AC) promotes pancreatic cancer (PC) progression and chemoresistance, suggesting its clinical association with poor prognosis. RNA sequencing analysis from the autochthonous pancreatic tumors showed a significant stromal alteration on genetic ablation of Muc5ac. Previously, depletion or targeting the stromal fibroblasts showed an ambiguous effect on PC pathogenesis. Hence, identifying the molecular players and mechanisms driving fibroblast heterogeneity is critical for improved clinical outcomes. METHODS: Autochthonous murine models of PC (KrasG12D, Pdx1-Cre [KC] and KrasG12D, Pdx1-Cre, Muc5ac-/- [KCM]) and co-implanted allografts of murine PC cell lines (Muc5ac wild-type and CRISPR/Cas knockout) with adipose-derived mesenchymal stem cells (AD-MSCs) were used to assess the role of Muc5ac in stromal heterogeneity. Proliferation, migration, and surface expression of cell-adhesion markers on AD-MSCs were measured using live-cell imaging and flow cytometry. MUC5AC-interactome was investigated using mass-spectrometry and enzyme-linked immunosorbent assay. RESULTS: The KCM tumors showed a significant decrease in the expression of α-smooth muscle actin and fibronectin compared with histology-matched KC tumors. Our study showed that MUC5AC, carrying tumor secretome, gets enriched in the adipose tissues of tumor-bearing mice and patients with PC, promoting CD44/CD29 (integrin-ß1) clustering that leads to Rac1 activation and migration of AD-MSCs. Furthermore, treatment with KC-derived serum enhanced proliferation and migration of AD-MSCs, which was abolished on Muc5ac-depletion or pharmacologic inhibition of CXCR2 and Rac1, respectively. The AD-MSCs significantly contribute toward α-smooth muscle actin-positive cancer-associated fibroblasts population in Muc5ac-dependent manner, as suggested by autochthonous tumors, co-implantation xenografts, and patient tumors. CONCLUSION: MUC5AC, secreted during PC progression, enriches in adipose and enhances the mobilization of AD-MSCs. On recruitment to pancreatic tumors, AD-MSCs proliferate and contribute towards stromal heterogeneity.
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Receptores de Hialuranos , Integrina beta1 , Células Madre Mesenquimatosas , Mucina 5AC , Neoplasias Pancreáticas , Actinas/metabolismo , Animales , Análisis por Conglomerados , Xenoinjertos , Humanos , Receptores de Hialuranos/metabolismo , Integrina beta1/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Mucina 5AC/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
BACKGROUND & AIMS: A major clinical challenge for patients with pancreatic cancer (PC) is metabolic adaptation. Neoplastic cells harboring molecular perturbations suffice for their increased anabolic demand and nucleotide biosynthesis to acquire chemoresistance. The mucin 5AC expressed de novo in malignant pancreas promotes cancer cell stemness and is significantly associated with poor patient survival. Identification of MUC5AC-associated drivers of chemoresistance through metabolic alterations may facilitate the sculpting of a new combinatorial regimen. METHODS: The contributions of MUC5AC to glutaminolysis and gemcitabine resistance were examined by The Cancer Genome Atlas data analysis, RNA sequencing, and immunohistochemistry analysis on pancreatic tissues of KrasG12D;Pdx1-Cre (KC) and KrasG12D;Pdx1-Cre;Muc5ac-/- mice. These were followed by metabolite flux assays as well as biochemical and xenograft studies on MUC5AC-depleted human and murine PC cells. Murine and human pancreatic 3-dimensional tumoroids were used to evaluate the efficacy of gemcitabine in combination with ß-catenin and glutaminolysis inhibitors. RESULTS: Transcriptional analysis showed that high MUC5AC-expressing human and autochthonous murine PC tumors exhibit higher resistance to gemcitabine because of enhanced glutamine use and nucleotide biosynthesis. Gemcitabine treatment led to MUC5AC overexpression, resulting in disruption of E-cadherin/ß-catenin junctions and the nuclear translocation of ß-catenin, which increased c-Myc expression, with a concomitant rise in glutamine uptake and glutamate release. MUC5AC depletion and glutamine deprivation sensitized human PC cells to gemcitabine, which was obviated by glutamine replenishment in MUC5AC-expressing cells. Coadministration of ß-catenin and glutaminolysis inhibitors with gemcitabine abrogated the MUC5AC-mediated resistance in murine and human tumoroids. CONCLUSIONS: The MUC5AC/ß-catenin/c-Myc axis increases the uptake and use of glutamine in PC cells, and cotargeting this axis along with gemcitabine may improve therapeutic efficacy in PC.
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Antimetabolitos Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos , Metabolismo Energético/efectos de los fármacos , Glutamina/metabolismo , Mucina 5AC/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular Tumoral , Bases de Datos Genéticas , Desoxicitidina/farmacología , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Glutaminasa/antagonistas & inhibidores , Glutaminasa/metabolismo , Humanos , Masculino , Ratones Noqueados , Ratones Desnudos , Mucina 5AC/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-myc/genética , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/antagonistas & inhibidores , beta Catenina/genética , GemcitabinaRESUMEN
BACKGROUND & AIMS: Epidemiological studies have established alcohol and smoking as independent risk factors for recurrent acute pancreatitis and chronic pancreatitis. However, the molecular players responsible for the progressive loss of pancreatic parenchyma and fibroinflammatory response are poorly characterized. METHODS: Tandem mass tag-based proteomic and bioinformatics analyses were performed on the pancreata of mice exposed to alcohol, cigarette smoke, or a combination of alcohol and cigarette smoke. Biochemical, immunohistochemistry, and transcriptome analyses were performed on the pancreatic tissues and primary acinar cells treated with cerulein in combination with ethanol (50 mmol/L) and cigarette smoke extract (40 µg/mL) for the mechanistic studies. RESULTS: A unique alteration in the pancreatic proteome was observed in mice exposed chronically to the combination of alcohol and cigarette smoke (56.5%) compared with cigarette smoke (21%) or alcohol (17%) alone. The formation of toxic metabolites (P < .001) and attenuated unfolded protein response (P < .04) were the significantly altered pathways on combined exposure. The extracellular matrix (ECM) proteins showed stable malondialdehyde-acetaldehyde (MAA) adducts in the pancreata of the combination group and chronic pancreatitis patients with a history of smoking and alcohol consumption. Interestingly, MAA-ECM adducts significantly suppressed expression of X-box-binding protein-1, leading to acinar cell death in the presence of alcohol and smoking. The stable MAA-ECM adducts persist even after alcohol and smoking cessation, and significantly delay pancreatic regeneration by abrogating the expression of cyclin-dependent kinases (CDK7 and CDK5) and regeneration markers. CONCLUSIONS: The combined alcohol and smoking generate stable MAA-ECM adducts that increase endoplasmic reticulum stress and acinar cell death due to attenuated unfolded protein response and suppress expression of cell cycle regulators. Targeting aldehyde adducts might provide a novel therapeutic strategy for the management of recurrent acute pancreatitis and chronic pancreatitis.
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Acetaldehído , Pancreatitis Crónica , Acetaldehído/metabolismo , Enfermedad Aguda , Aldehídos , Animales , Ceruletida , Quinasas Ciclina-Dependientes/metabolismo , Etanol/toxicidad , Proteínas de la Matriz Extracelular/metabolismo , Malondialdehído/metabolismo , Ratones , Proteoma/metabolismo , Proteómica , Fumar/efectos adversos , Respuesta de Proteína DesplegadaRESUMEN
Myeloid derived suppressor cells (MDSCs) can be subset into monocytic (M-), granulocytic (G-) or polymorphonuclear (PMN-), and immature (i-) or early MDSCs and have a role in many disease states. In cancer patients, the frequencies of MDSCs can positively correlate with stage, grade, and survival. Most clinical studies into MDSCs have been undertaken with peripheral blood (PB); however, in the present studies, we uniquely examined MDSCs in the spleens and PB from patients with gastrointestinal cancers. In our studies, MDSCs were rigorously subset using the following markers: Lineage (LIN) (CD3, CD19 and CD56), human leukocyte antigen (HLA)-DR, CD11b, CD14, CD15, CD33, CD34, CD45, and CD16. We observed a significantly higher frequency of PMN- and M-MDSCs in the PB of cancer patients as compared to their spleens. Expression of the T-cell suppressive enzymes arginase (ARG1) and inducible nitric oxide synthase (i-NOS) were higher on all MDSC subsets for both cancer patients PB and spleen cells as compared to MDSCs from the PB of normal donors. Similar findings for the activation markers lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), program death ligand 1 (PD-L1) and program cell death protein 1 (PD-1) were observed. Interestingly, the total MDSC cell number exported to clustering analyses was similar between all sample types; however, clustering analyses of these MDSCs, using these markers, uniquely documented novel subsets of PMN-, M- and i-MDSCs. In summary, we report a comparison of splenic MDSC frequency, subtypes, and functionality in cancer patients to their PB by clustering and cytometric analyses.
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Células Supresoras de Origen Mieloide/metabolismo , Bazo/inmunología , Adulto , Anciano , Arginasa/metabolismo , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Análisis por Conglomerados , Femenino , Citometría de Flujo/métodos , Neoplasias Gastrointestinales/inmunología , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Células Supresoras de Origen Mieloide/citología , Células Supresoras de Origen Mieloide/inmunología , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Receptores Depuradores de Clase E/metabolismo , Bazo/patologíaRESUMEN
BACKGROUND: Differential expression of mucins has been associated with several cancers including colorectal cancer (CRC). In normal physiological conditions, secretory mucin MUC5AC is not expressed in the colonic mucosa, whereas its aberrant expression is observed during development of colon cancer and its precursor lesions. To date, the molecular mechanism of MUC5AC in CRC progression and drug resistance remains obscure. METHODS: MUC5AC expression was determined in colon tissue microarray by immunohistochemistry. A RNA interference and CRISPR/Cas9-mediated system was used to knockdown/knockout the MUC5AC in CRC cell lines to delineate its role in CRC tumorigenesis using in vitro functional assays and in vivo (sub-cutaneous and colon orthotopic) mouse models. Finally, CRC cell lines and xenograft models were used to identify the mechanism of action of MUC5AC. RESULTS: Overexpression of MUC5AC is observed in CRC patient tissues and cell lines. MUC5AC expression resulted in enhanced cell invasion and migration, and decreased apoptosis of CRC cells. MUC5AC interacted with CD44 physically, which was accompanied by the activation of Src signaling. Further, the presence of MUC5AC resulted in enhanced tumorigenesis and appearance of metastatic lesions in orthotopic mouse model. Additionally, up-regulation of MUC5AC resulted in resistance to 5-fluorouracil (5-FU) and oxaliplatin, and its knockout increased sensitivity to these drugs. Finally, we observed that up-regulation of MUC5AC conferred resistance to 5-FU through down-regulation of p53 and its target gene p21 and up-regulation of ß-catenin and its target genes CD44 and Lgr5. CONCLUSION: Our findings suggest that differential expression of secretory mucin MUC5AC results in enhanced tumorigenesis and also confers chemoresistance via CD44/ß-catenin/p53/p21 signaling.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/patología , Resistencia a Antineoplásicos , Receptores de Hialuranos/metabolismo , Mucina 5AC/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Progresión de la Enfermedad , Fluorouracilo/administración & dosificación , Regulación Neoplásica de la Expresión Génica , Humanos , Receptores de Hialuranos/genética , Ratones , Ratones Desnudos , Mucina 5AC/genética , Oxaliplatino/administración & dosificación , Pronóstico , Tasa de Supervivencia , Células Tumorales Cultivadas , Vía de Señalización Wnt , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND: Quiescent tumor cells pose a major clinical challenge due to their ability to resist conventional chemotherapies and to drive tumor recurrence. Understanding the molecular mechanisms that promote quiescence of tumor cells could help identify therapies to eliminate these cells. Significantly, recent studies have determined that the function of SOX2 in cancer cells is highly dose dependent. Specifically, SOX2 levels in tumor cells are optimized to promote tumor growth: knocking down or elevating SOX2 inhibits proliferation. Furthermore, recent studies have shown that quiescent tumor cells express higher levels of SOX2 compared to adjacent proliferating cells. Currently, the mechanisms through which elevated levels of SOX2 restrict tumor cell proliferation have not been characterized. METHODS: To understand how elevated levels of SOX2 restrict the proliferation of tumor cells, we engineered diverse types of tumor cells for inducible overexpression of SOX2. Using these cells, we examined the effects of elevating SOX2 on their proliferation, both in vitro and in vivo. In addition, we examined how elevating SOX2 influences their expression of cyclins, cyclin-dependent kinases (CDKs), and p27Kip1. RESULTS: Elevating SOX2 in diverse tumor cell types led to growth inhibition in vitro. Significantly, elevating SOX2 in vivo in pancreatic ductal adenocarcinoma, medulloblastoma, and prostate cancer cells induced a reversible state of tumor growth arrest. In all three tumor types, elevation of SOX2 in vivo quickly halted tumor growth. Remarkably, tumor growth resumed rapidly when SOX2 returned to endogenous levels. We also determined that elevation of SOX2 in six tumor cell lines decreased the levels of cyclins and CDKs that control each phase of the cell cycle, while upregulating p27Kip1. CONCLUSIONS: Our findings indicate that elevating SOX2 above endogenous levels in a diverse set of tumor cell types leads to growth inhibition both in vitro and in vivo. Moreover, our findings indicate that SOX2 can function as a master regulator by controlling the expression of a broad spectrum of cell cycle machinery. Importantly, our SOX2-inducible tumor studies provide a novel model system for investigating the molecular mechanisms by which elevated levels of SOX2 restrict cell proliferation and tumor growth.
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Proliferación Celular/genética , Recurrencia Local de Neoplasia/genética , Neoplasias/genética , Factores de Transcripción SOXB1/genética , Apoptosis/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/genética , Ciclinas/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Recurrencia Local de Neoplasia/patología , Neoplasias/patologíaRESUMEN
Therapeutic plasma exchange (TPE) has been demonstrated to be of significant clinical value in a number of diseases and conditions, with well-established guidelines and recommendations. However, technical support in providing this procedure for pregnant patients is largely absent from these recommendations, leaving therapeutic apheresis practitioners without guidance to safely and adequately treat appropriate conditions in this important patient population. Here, we describe our experience in treating a 35-year-old pregnant patient with relapsing-remitting multiple sclerosis with TPE. Additionally, we outline the principle considerations when developing her treatment plan, and we provide recommendations for apheresis practitioners when performing TPE in pregnant patients. J. Clin. Apheresis 32:191-195, 2017. © 2016 Wiley Periodicals, Inc.
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Esclerosis Múltiple Recurrente-Remitente/terapia , Intercambio Plasmático/normas , Adulto , Femenino , Humanos , Guías de Práctica Clínica como Asunto , Embarazo , Resultado del TratamientoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to limited therapeutic options and expensive/burdensome drug discovery processes. Utilizing genomic-data-driven Connectivity Mapping (CMAP) to identify a drug closer to real-world PC targeting may improve pancreatic cancer (PC) patient outcomes. Initially, we mapped CMAP data to gene expression from 106 PC patients, identifying nine negatively connected drugs. These drugs were further narrowed down using a similar analysis for PC cell lines, human tumoroids, and patient-derived xenografts datasets, where ISOX emerged as the most potent agent to target PC. We used human and mouse syngeneic PC cells, human and mouse tumoroids, and in vivo mice to assess the ability of ISOX alone and in combination with 5FU to inhibit tumor growth. Global transcriptomic and pathway analysis of the ISOX-LINCS signature identified HDAC 6/cMyc as the target axis for ISOX. Specifically, we discovered that genetic and pharmacological targeting of HDAC 6 affected non-histone protein cMyc acetylation, leading to cMyc instability, thereby disrupting PC growth and metastasis by affecting cancer stemness. Finally, KrasG12D harboring tumoroids and mice responded effectively against ISOX and 5FU treatment by enhancing survival and controlling metastasis incidence. Overall, our data validate ISOX as a new drug to treat advanced PC patients without toxicity to normal cells. Our study supports the clinical utility of ISOX along with 5FU in future PC clinical trials.
RESUMEN
Gemcitabine is the first-line treatment option for patients with locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, the frequent adoption of resistance to gemcitabine by cancer cells poses a significant challenge in treating this aggressive disease. In this study, we focused on analyzing the role of trefoil factor 1 (TFF1) in gemcitabine resistance in PDAC. Analysis of PDAC TCGA and cell line datasets indicated an enrichment of TFF1 in the gemcitabine-resistant classical subtype and suggested an inverse correlation between TFF1 expression and sensitivity to gemcitabine treatment. The genetic ablation of TFF1 in PDAC cells enhanced their sensitivity to gemcitabine treatment in both in vitro and in vivo tumor xenografts. The biochemical studies revealed that TFF1 contributes to gemcitabine resistance through enhanced stemness, increasing migration ability of cancer cells, and induction of anti-apoptotic genes. We further pursued studies to predict possible receptors exerting TFF1-mediated gemcitabine resistance. Protein-protein docking investigations with BioLuminate software revealed that TFF1 binds to the chemokine receptor CXCR4, which was supported by real-time binding analysis of TFF1 and CXCR4 using SPR studies. The exogenous addition of TFF1 increased the proliferation and migration of PDAC cells through the pAkt/pERK axis, which was abrogated by treatment with a CXCR4-specific antagonist AMD3100. Overall, the present study demonstrates the contribution of the TFF1-CXCR4 axis in imparting gemcitabine resistance properties to PDAC cells.
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Antimetabolitos Antineoplásicos , Carcinoma Ductal Pancreático , Desoxicitidina , Resistencia a Antineoplásicos , Gemcitabina , Neoplasias Pancreáticas , Receptores CXCR4 , Factor Trefoil-1 , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Factor Trefoil-1/genética , Factor Trefoil-1/metabolismo , Animales , Línea Celular Tumoral , Antimetabolitos Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Ratones , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Ratones Desnudos , Proliferación Celular/efectos de los fármacos , Simulación del Acoplamiento MolecularRESUMEN
Unbiased proteomic screens provide a powerful tool for defining protein-protein interaction networks. Previous studies employed multidimensional protein identification technology to identify the Sox2-interactome in embryonic stem cells (ESC) undergoing differentiation in response to a small increase in the expression of epitope-tagged Sox2. Thus far the Sox2-interactome in ESC has not been determined. To identify the Sox2-interactome in ESC, we engineered ESC for inducible expression of different combinations of epitope-tagged Sox2 along with Oct4, Klf4, and c-Myc. Epitope-tagged Sox2 was used to circumvent the lack of suitable Sox2 antibodies needed to perform an unbiased proteomic screen of Sox2-associated proteins. Although i-OS-ESC differentiate when both Oct4 and Sox2 are elevated, i-OSKM-ESC do not differentiate even when the levels of the four transcription factors are coordinately elevated â¼2-3-fold. Our findings with i-OS-ESC and i-OSKM-ESC provide new insights into the reasons why ESC undergo differentiation when Sox2 and Oct4 are elevated in ESC. Importantly, the use of i-OSKM-ESC enabled us to identify the Sox2-interactome in undifferentiated ESC. Using multidimensional protein identification technology, we identified >70 proteins that associate with Sox2 in ESC. We extended these findings by testing the function of the Sox2-assoicated protein Smarcd1 and demonstrate that knockdown of Smarcd1 disrupts the self-renewal of ESC and induces their differentiation. Together, our work provides the first description of the Sox2-interactome in ESC and indicates that Sox2 along with other master regulators is part of a highly integrated protein-protein interaction landscape in ESC.
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Ingeniería Celular , Células Madre Embrionarias/metabolismo , Mapeo de Interacción de Proteínas , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Animales , Diferenciación Celular , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Células Madre Embrionarias/citología , Mapeo Epitopo , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Factor 4 Similar a Kruppel , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Proteómica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
Self-renewal is a complex biological process necessary for maintaining the pluripotency of embryonic stem cells (ESCs). Recent studies have used global proteomic techniques to identify proteins that associate with the master regulators Oct4, Nanog and Sox2 in ESCs or in ESCs during the early stages of differentiation. Through an unbiased proteomic screen, Banf1 was identified as a Sox2-associated protein. Banf1 has been shown to be essential for worm and fly development but, until now, its role in mammalian development and ESCs has not been explored. In this study, we examined the effect of knocking down Banf1 on ESCs. We demonstrate that the knockdown of Banf1 promotes the differentiation of mouse ESCs and decreases the survival of both mouse and human ESCs. For mouse ESCs, we demonstrate that knocking down Banf1 promotes their differentiation into cells that exhibit markers primarily associated with mesoderm and trophectoderm. Interestingly, knockdown of Banf1 disrupts the survival of human ESCs without significantly reducing the expression levels of the master regulators Sox2, Oct4 and Nanog or inducing the expression of markers of differentiation. Furthermore, we determined that the knockdown of Banf1 alters the cell cycle distribution of both human and mouse ESCs by causing an uncharacteristic increase in the proportion of cells in the G2-M phase of the cell cycle.
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Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas Nucleares/metabolismo , Animales , Western Blotting , Ciclo Celular/genética , Ciclo Celular/fisiología , Línea Celular , Proteínas de Unión al ADN/genética , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Ratones , Proteínas Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
PURPOSE: Despite the significant association of molecular subtypes with poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC), few efforts have been made to identify the underlying pathway(s) responsible for this prognosis. Identifying a clinically relevant prognosis-based gene signature may be the key to improving patient outcomes. EXPERIMENTAL DESIGN: We analyzed the transcriptomic profiles of treatment-naïve surgically resected short-term survivor (STS) and long-term survivor (LTS) tumors (GSE62452) for expression and survival, followed by validation in several datasets. These results were corroborated by IHC analysis of PDAC-resected STS and LTS tumors. The mechanism of this differential survival was investigated using CIBERSORT and pathway analyses. RESULTS: We identified a short-surviving prognostic subtype of PDAC with a high degree of significance (P = 0.018). One hundred thirty genes in this novel subtype were found to be regulated by a master regulator, homeobox gene HOXA10, and a 5-gene signature derived from these genes, including BANF1, EIF4G1, MRPS10, PDIA4, and TYMS, exhibited differential expression in STSs and a strong association with poor survival. This signature was further associated with the proportion of T cells and macrophages found in STSs and LTSs, demonstrating a potential role in PDAC immunosuppression. Pathway analyses corroborated these findings, revealing that this HOXA10-driven prognostic signature is associated with immune suppression and enhanced tumorigenesis. CONCLUSIONS: Overall, these findings reveal the presence of a HOXA10-associated prognostic subtype that can be used to differentiate between STS and LTS patients of PDAC and inform on the molecular interactions that play a role in this poor prognosis.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Pronóstico , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Transcriptoma , Regulación Neoplásica de la Expresión Génica , Proteínas Homeobox A10/genética , Proteínas Homeobox A10/metabolismoRESUMEN
Mucin4 (MUC4) appears early during pancreatic intraepithelial neoplasia-1 (PanIN1), coinciding with the expression of epidermal growth factor receptor-1 (EGFR). The EGFR signaling is required for the onset of Kras-driven pancreatic ductal adenocarcinoma (PDAC); however, the players and mechanisms involved in sustained EGFR signaling in early PanIN lesions remain elusive. We generated a unique Esai-CRISPR-based Muc4 conditional knockout murine model to evaluate its effect on PDAC pathology. The Muc4 depletion in the autochthonous murine model carrying K-ras and p53 mutations (K-rasG12D; TP53R172H; Pdx-1cre, KPC) to generate the KPCM4-/- murine model showed a significant delay in the PanIN lesion formation with a significant reduction (p < 0.01) in EGFR (Y1068) and ERK1/2 (T202/Y204) phosphorylation. Further, a significant decrease (p < 0.01) in Sox9 expression in PanIN lesions of KPCM4-/- mice suggested the impairment of acinar-to-ductal metaplasia in Muc4-depleted cells. The biochemical analyses demonstrated that MUC4, through its juxtamembrane EGF-like domains, interacts with the EGFR ectodomain, and its cytoplasmic tail prevents EGFR ubiquitination and subsequent proteasomal degradation upon ligand stimulation, leading to sustained downstream oncogenic signaling. Targeting the MUC4 and EGFR interacting interface provides a promising strategy to improve the efficacy of EGFR-targeted therapies in PDAC and other MUC4-expressing malignancies.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ratones , Animales , Fosforilación , Modelos Animales de Enfermedad , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Transformación Celular Neoplásica/genética , Carcinogénesis , Receptores ErbB/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Neoplasias PancreáticasRESUMEN
Cisplatin- and gemcitabine-based chemotherapeutics represent a mainstay of cancer therapy for most solid tumors; however, resistance limits their curative potential. Here, we identify RNA polymerase II-associated factor 1 (PAF1) as a common driver of cisplatin and gemcitabine resistance in human cancers (ovarian, lung, and pancreas). Mechanistically, cisplatin- and gemcitabine-resistant cells show enhanced DNA repair, which is inhibited by PAF1 silencing. We demonstrate an increased interaction of PAF1 with RAD52 in resistant cells. Targeting the PAF1 and RAD52 axis combined with cisplatin or gemcitabine strongly diminishes the survival potential of resistant cells. Overall, this study shows clinical evidence that the expression of PAF1 contributes to chemotherapy resistance and worse clinical outcome for lethal cancers.
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Carcinoma de Pulmón de Células no Pequeñas , Resistencia a Antineoplásicos , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Cisplatino/uso terapéutico , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Gemcitabina/uso terapéutico , Neoplasias Pulmonares/genética , Proteína Recombinante y Reparadora de ADN Rad52 , Factores de TranscripciónRESUMEN
Aberrantly expressed onco-mucin 16 (MUC16) and its post-cleavage generated surface tethered carboxy-terminal (MUC16-Cter) domain are strongly associated with poor prognosis and lethality of pancreatic (PC) and non-small cell lung cancer (NSCLC). To date, most anti-MUC16 antibodies are directed towards the extracellular domain of MUC16 (CA125), which is usually cleaved and shed in the circulation hence obscuring antibody accessibility to the cancer cells. Herein, we establish the utility of targeting a post-cleavage generated, surface-tethered oncogenic MUC16 carboxy-terminal (MUC16-Cter) domain by using a novel chimeric antibody in human IgG1 format, ch5E6, whose epitope expression directly correlates with disease severity in both cancers. ch5E6 binds and interferes with MUC16-associated oncogenesis, suppresses the downstream signaling pFAK(Y397)/p-p70S6K(T389)/N-cadherin axis and exert antiproliferative effects in cancer cells, 3D organoids, and tumor xenografts of both PC and NSCLC. The robust clinical correlations observed between MUC16 and N-cadherin in patient tumors and metastatic samples imply ch5E6 potential in targeting a complex and significantly occurring phenomenon of epithelial to mesenchymal transition (EMT) associated with disease aggressiveness. Our study supports evaluating ch5E6 with standard-of-care drugs, to potentially augment treatment outcomes in malignancies inflicted with MUC16-associated poor prognosis.
RESUMEN
Ras family proteins are membrane-bound GTPases that control proliferation, survival, and motility. Many forms of cancers are driven by the acquisition of somatic mutations in a RAS gene. In pancreatic cancer (PC), more than 90% of tumors carry an activating mutation in KRAS. Mutations in components of the Ras signaling pathway can also be the cause of RASopathies, a group of developmental disorders. In a subset of RASopathies, the causal mutations are in the LZTR1 protein, a substrate adaptor for E3 ubiquitin ligases that promote the degradation of Ras proteins. Here, we show that the function of LZTR1 is regulated by the glycogen synthase kinase 3 (GSK3). In PC cells, inhibiting or silencing GSK3 led to a decline in the level of Ras proteins, including both wild type Ras proteins and the oncogenic Kras protein. This decline was accompanied by a 3-fold decrease in the half-life of Ras proteins and was blocked by the inhibition of the proteasome or the knockdown of LZTR1. Irrespective of the mutational status of KRAS, the decline in Ras proteins was observed and accompanied by a loss of cell proliferation. This loss of proliferation was blocked by the knockdown of LZTR1 and could be recapitulated by the silencing of either KRAS or GSK3. These results reveal a novel GSK3-regulated LZTR1-dependent mechanism that controls the stability of Ras proteins and proliferation of PC cells. The significance of this novel pathway to Ras signaling and its contribution to the therapeutic properties of GSK3 inhibitors are both discussed.
Asunto(s)
Glucógeno Sintasa Quinasa 3 , Neoplasias Pancreáticas , Proliferación Celular/genética , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Neoplasias Pancreáticas/genética , Transducción de Señal , Factores de Transcripción/genética , Proteínas ras/metabolismoRESUMEN
Delivery of therapeutic agents in pancreatic cancer (PC) is impaired due to its hypovascular and desmoplastic tumor microenvironment. The Endothelin (ET)-axis is the major regulator of vasomotor tone under physiological conditions and is highly upregulated in multiple cancers. We investigated the effect of dual endothelin receptor antagonist bosentan on perfusion and macromolecular transport in a PC cell-fibroblast co-implantation tumor model using Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI). Following bosentan treatment, the contrast enhancement ratio and wash-in rates in tumors were two- and nine times higher, respectively, compared to the controls, whereas the time to peak was significantly shorter (7.29 ± 1.29 min v/s 22.08 ± 5.88 min; p = 0.04). Importantly, these effects were tumor selective as the magnitudes of change for these parameters were much lower in muscles. Bosentan treatment also reduced desmoplasia and improved intratumoral distribution of high molecular weight FITC-dextran. Overall, these findings support that targeting the ET-axis can serve as a potential strategy to selectively enhance tumor perfusion and improve the delivery of therapeutic agents in pancreatic tumors.
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Antagonistas de los Receptores de Endotelina , Neoplasias Pancreáticas , Bosentán , Antagonistas de los Receptores de Endotelina/farmacología , Antagonistas de los Receptores de Endotelina/uso terapéutico , Endotelinas , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Perfusión , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
In recent years, immune therapy, notably immune checkpoint inhibitors (ICI), in conjunction with chemotherapy and surgery has demonstrated therapeutic activity for some tumor types. However, little is known about the optimal combination of immune therapy with standard of care therapies and approaches. In patients with gastrointestinal (GI) cancers, especially pancreatic ductal adenocarcinoma (PDAC), preoperative (neoadjuvant) chemotherapy has increased the number of patients who can undergo surgery and improved their responses. However, most chemotherapy is immunosuppressive, and few studies have examined the impact of neoadjuvant chemotherapy (NCT) on patient immunity and/or the optimal combination of chemotherapy with immune therapy. Furthermore, the majority of chemo/immunotherapy studies focused on immune regulation in cancer patients have focused on postoperative (adjuvant) chemotherapy and are limited to peripheral blood (PB) and occasionally tumor infiltrating lymphocytes (TILs); representing a minority of immune cells in the host. Our previous studies examined the phenotype and frequencies of myeloid and lymphoid cells in the PB and spleens of GI cancer patients, independent of chemotherapy regimen. These results led us to question the impact of NCT on host immunity. We report herein, unique studies examining the splenic and PB phenotypes, frequencies, and numbers of myeloid and lymphoid cell populations in NCT treated GI cancer patients, as compared to treatment naïve cancer patients and patients with benign GI tumors at surgery. Overall, we noted limited immunological differences in patients 6 weeks following NCT (at surgery), as compared to treatment naive patients, supporting rapid immune normalization. We observed that NCT patients had a lower myeloid derived suppressor cells (MDSCs) frequency in the spleen, but not the PB, as compared to treatment naive cancer patients and patients with benign GI tumors. Further, NCT patients had a higher splenic and PB frequency of CD4+ T-cells, and checkpoint protein expression, as compared to untreated, cancer patients and patients with benign GI tumors. Interestingly, in NCT treated cancer patients the frequency of mature (CD45RO+) CD4+ and CD8+ T-cells in the PB and spleens was higher than in treatment naive patients. These differences may also be associated, in part with patient stage, tumor grade, and/or NCT treatment regimen. In summary, the phenotypic profile of leukocytes at the time of surgery, approximately 6 weeks following NCT treatment in GI cancer patients, are similar to treatment naive GI cancer patients (i.e., patients who receive adjuvant therapy); suggesting that NCT may not limit the response to immune intervention and may improve tumor responses due to the lower splenic frequency of MDSCs and higher frequency of mature T-cells.
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Neoplasias Gastrointestinales , Neoplasias Pancreáticas , Linfocitos T CD8-positivos , Neoplasias Gastrointestinales/tratamiento farmacológico , Humanos , Terapia Neoadyuvante , Neoplasias Pancreáticas/patología , BazoRESUMEN
Acinar-to-ductal metaplasia (ADM) is a precursor lesion of pancreatic ductal adenocarcinoma (PDAC); however, the regulators of the ADM-mediated PDAC development and its targeting are poorly understood. RNA polymerase II-associated factor 1 (PAF1) maintains cancer stem cells leading to the aggressiveness of PDAC. In this study, we investigated whether PAF1 is required for the YAP1-mediated PDAC development and whether CA3 and verteporfin, small molecule inhibitors of YAP1/TEAD transcriptional activity, diminish pancreatic cancer (PC) cell growth by targeting the PAF1/YAP1 axis. Here, we demonstrated that PAF1 co-expresses and interacts with YAP1 specifically in metaplastic ducts of mouse cerulein- or KrasG12D-induced ADM and human PDAC but not in the normal pancreas. PAF1 knockdown (KD) reduced SOX9 in PC cells, and the PC cells showed elevated PAF1/YAP1 complex recruitment to the promoter of SOX9. The PAF1 KD reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in the mouse KC (KrasG12D; Pdx-1 Cre) cells and human PC cells, indicating that the PAF1 is required for the YAP1-mediated development of ADM and PC. Moreover, treatment with CA3 or verteporfin reduced the expressions of PAF1, YAP1, TEAD4, and SOX9 and decreased colony formation and stemness in KC and PC cells. CA3 treatment also reduced the viability and proliferation of PC cells and diminished the duct-like structures in KC acinar explants. CA3 or verteporfin treatment decreased the recruitment of the PAF1/YAP1 complex to the SOX9 promoter in PC cells and reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in KC and PC cells. Overall, PAF1 cooperates with YAP1 during ADM and PC development, and verteporfin and CA3 inhibit ADM and PC cell growth by targeting the PAF1/YAP1/SOX9 axis in vitro and ex vivo models. This study identified a regulatory axis of PDAC initiation and its targeting, paving the way for developing targeted therapeutic strategies for pancreatic cancer patients.