RESUMEN
BACKGROUND: Small cell lung cancer (SCLC) is an aggressive lung cancer subtype that is associated with high recurrence and poor prognosis. Due to lack of potential drug targets, SCLC patients have few therapeutic options. MicroRNAs (miRNAs) provide an interesting repertoire of therapeutic molecules; however, the identification of miRNAs regulating SCLC growth and metastasis and their precise regulatory mechanisms are not well understood. METHODS: To identify novel miRNAs regulating SCLC, we performed miRNA-sequencing from donor/patient serum samples and analyzed the bulk RNA-sequencing data from the tumors of SCLC patients. Further, we developed a nanotechnology-based, highly sensitive method to detect microRNA-1 (miR-1, identified miRNA) in patient serum samples and SCLC cell lines. To assess the therapeutic potential of miR-1, we developed various in vitro models, including miR-1 sponge (miR-1Zip) and DOX-On-miR-1 (Tet-ON) inducible stable overexpression systems. Mouse models derived from intracardiac injection of SCLC cells (miR-1Zip and DOX-On-miR-1) were established to delineate the role of miR-1 in SCLC metastasis. In situ hybridization and immunohistochemistry were used to analyze the expression of miR-1 and target proteins (mouse and human tumor specimens), respectively. Dual-luciferase assay was used to validate the target of miR-1, and chromatin immunoprecipitation assay was used to investigate the protein-gene interactions. RESULTS: A consistent downregulation of miR-1 was observed in tumor tissues and serum samples of SCLC patients compared to their matched normal controls, and these results were recapitulated in SCLC cell lines. Gain of function studies of miR-1 in SCLC cell lines showed decreased cell growth and oncogenic signaling, whereas loss of function studies of miR-1 rescued this effect. Intracardiac injection of gain of function of miR-1 SCLC cell lines in the mouse models showed a decrease in distant organ metastasis, whereas loss of function of miR-1 potentiated growth and metastasis. Mechanistic studies revealed that CXCR4 is a direct target of miR-1 in SCLC. Using unbiased transcriptomic analysis, we identified CXCR4/FOXM1/RRM2 as a unique axis that regulates SCLC growth and metastasis. Our results further showed that FOXM1 directly binds to the RRM2 promoter and regulates its activity in SCLC. CONCLUSIONS: Our findings revealed that miR-1 is a critical regulator for decreasing SCLC growth and metastasis. It targets the CXCR4/FOXM1/RRM2 axis and has a high potential for the development of novel SCLC therapies. MicroRNA-1 (miR-1) downregulation in the tumor tissues and serum samples of SCLC patients is an important hallmark of tumor growth and metastasis. The introduction of miR-1 in SCLC cell lines decreases cell growth and metastasis. Mechanistically, miR-1 directly targets CXCR4, which further prevents FOXM1 binding to the RRM2 promoter and decreases SCLC growth and metastasis.
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Neoplasias Pulmonares , MicroARNs , Carcinoma Pulmonar de Células Pequeñas , Humanos , Animales , Ratones , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , MicroARNs/genética , MicroARNs/metabolismo , Línea Celular Tumoral , Neoplasias Pulmonares/patología , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Proteína Forkhead Box M1/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismoRESUMEN
There is a major need for the development of new therapeutics to combat antibiotic-resistant Staphylococcus aureus. Recently, gallium (Ga)-based complexes have shown promising antimicrobial effects against various bacteria, including multidrug-resistant organisms, by targeting multiple heme/iron-dependent metabolic pathways. Among these, Ga protoporphyrin (GaPP) inhibits bacterial growth by targeting heme pathways, including aerobic respiration. Ga(NO3)3, an iron mimetic, disrupts elemental iron pathways. Here, we demonstrate the enhanced antimicrobial activity of the combination of GaPP and Ga(NO3)3 against methicillin-resistant S. aureus (MRSA) under iron-limited conditions, including small colony variants (SCV). This therapy demonstrated significant antimicrobial activity without inducing slow-growing SCV. We also observed that the combination of GaPP and Ga(NO3)3 inhibited the MRSA catalase but not above that seen with Ga(NO3)3 alone. Neither GaPP nor Ga(NO3)3 alone or their combination inhibited the dominant superoxide dismutase expressed (SodA) under the iron-limited conditions examined. Intranasal administration of the combination of the two compounds improved drug biodistribution in the lungs compared to intraperitoneal administration. In a murine MRSA lung infection model, we observed a significant increase in survival and decrease in MRSA lung CFUs in mice that received combination therapy with intranasal GaPP and Ga(NO3)3 compared to untreated control or mice receiving GaPP or Ga(NO3)3 alone. No drug-related toxicity was observed as assessed histologically in the spleen, lung, nasal cavity, and kidney for both single and repeated doses of 10 mg Ga /Kg of mice over 13 days. Our results strongly suggest that GaPP and Ga(NO3)3 in combination have excellent synergism and potential to be developed as a novel therapy for infections with S. aureus.
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Galio , Staphylococcus aureus Resistente a Meticilina , Animales , Ratones , Protoporfirinas/farmacología , Protoporfirinas/metabolismo , Staphylococcus aureus , Distribución Tisular , Antibacterianos/farmacología , Galio/farmacología , Hemo/metabolismo , Hierro/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
BACKGROUND: A pro-oxidant enzyme, NADPH oxidase 4 (Nox4) has been reported to be a critical downstream effector of TGFß-induced myofibroblast transformation during fibrosis. While there are a small number of studies suggesting an oncogenic role of Nox4 derived from activated fibroblasts, direct evidence linking this pro-oxidant to the tumor-supporting CAF phenotype and the mechanisms involved are lacking, particularly in breast cancer. METHODS: We targeted Nox4 in breast patient-derived CAFs via siRNA-mediated knockdown or administration of a pharmaceutical inhibitor (GKT137831). We also determine primary tumor growth and metastasis of implanted tumor cells using a stable Nox4-/- syngeneic mouse model. Autophagic flux of CAFs was assessed using a tandem fluorescent-tagged ptfl-LC3 plasmid via confocal microscopy analysis and determination of the expression level of autophagy markers (beclin-1 and LC3B). Nox4 overexpressing CAFs depend on the Nrf2 (nuclear factor-erythroid factor 2-related factor 2) pathway for survival. We then determined the dependency of Nox4-overexpressing CAFs on the Nrf2-mediated adaptive stress response pathway for survival. Furthermore, we investigated the involvement of Birc5 on CAF phenotype (viability and collagen contraction activity) as well as the expression level of CAF markers, FAP and αSMA. CONCLUSIONS: We found that deletion of stroma Nox4 and pharmaceutically targeting its activity with GKT137831 significantly inhibited orthotopic tumor growth and metastasis of implanted E0771 and 4T1 murine mammary carcinoma cell lines in mice. More importantly, we found a significant upregulation of Nox4 expression in CAFs isolated from human breast tumors versus normal mammary fibroblasts (RMFs). Our in situ RNA hybridization analysis for Nox4 transcription on a human breast tumor microarray further support a role of this pro-oxidant in the stroma of breast carcinomas. In addition, we found that Nox4 promotes autophagy in CAFs. Moreover, we found that Nox4 promoted survival of CAFs via activation of Nrf2, a master regulator of oxidative stress response. We have further shown Birc5 is involved as a downstream modulator of Nrf2-mediated pro-survival phenotype. Together these studies indicate a role of redox signaling via the Nox4-Nrf2 pathway in tumorigenesis and metastasis of breast cancer cells by promoting autophagy and survival of CAFs.
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Neoplasias de la Mama , Fibroblastos Asociados al Cáncer , Animales , Neoplasias de la Mama/patología , Fibroblastos Asociados al Cáncer/patología , Carcinogénesis/metabolismo , Línea Celular Tumoral , Femenino , Fibroblastos/metabolismo , Humanos , Ratones , NADPH Oxidasa 4/genética , NADPH Oxidasa 4/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Survivin/metabolismo , Regulación hacia ArribaRESUMEN
Surgery remains the only potentially curative treatment option for pancreatic cancer, but resections are made more difficult by infiltrative disease, proximity of critical vasculature, peritumoral inflammation, and dense stroma. Surgeons are limited to tactile and visual cues to differentiate cancerous tissue from normal tissue. Furthermore, translating preoperative images to the intraoperative setting poses additional challenges for tumor detection, and can result in undetected and unresected lesions. Thus, pancreatic ductal adenocarcinoma (PDAC) has high rates of incomplete resections, and subsequently, disease recurrence. Fluorescence-guided surgery (FGS) has emerged as a method to improve intraoperative detection of cancer and ultimately improve surgical outcomes. Initial clinical trials have demonstrated feasibility of FGS for PDAC, but there are limited targeted probes under investigation for this disease, highlighting the need for development of additional novel biomarkers to reflect the PDAC heterogeneity. MUCIN16 (MUC16) is a glycoprotein that is overexpressed in 60-80% of PDAC. In our previous work, we developed a MUC16-targeted murine antibody near-infrared conjugate, termed AR9.6-IRDye800, that showed efficacy in detecting pancreatic cancer. To build on the translational potential of this imaging probe, a humanized variant of the AR9.6 fluorescent conjugate was developed and investigated herein. This conjugate, termed huAR9.6-IRDye800, showed equivalent binding properties to its murine counterpart. Using an optimized dye:protein ratio of 1:1, in vivo studies demonstrated high tumor to background ratios in MUC16-expressing tumor models, and delineation of tumors in a patient-derived xenograft model. Safety, biodistribution, and toxicity studies were conducted. These studies demonstrated that huAR9.6-IRDye800 was safe, did not yield evidence of histological toxicity, and was well tolerated in vivo. The results from this work suggest that AR9.6-IRDye800 is an efficacious and safe imaging agent for identifying pancreatic cancer intraoperatively through fluorescence-guided surgery.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Antígeno Ca-125/metabolismo , Carcinoma Ductal Pancreático/diagnóstico por imagen , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/cirugía , Línea Celular Tumoral , Colorantes Fluorescentes/química , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Recurrencia Local de Neoplasia , Imagen Óptica/métodos , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/cirugía , Distribución Tisular , Neoplasias PancreáticasRESUMEN
Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthetic pathway (IBP), produces the isoprenoid (geranylgeranyl pyrophosphate, GGPP) used in protein geranylgeranylation reactions. Our prior studies utilizing triazole bisphosphonate-based GGDPS inhibitors (GGSIs) have revealed that these agents represent a novel strategy by which to induce cancer cell death, including multiple myeloma and pancreatic cancer. Statins inhibit the rate-limiting enzyme in the IBP and potentiate the effects of GGSIs in vitro. The in vivo effects of combination therapy with statins and GGSIs have not been determined. Here we evaluated the effects of combining VSW1198, a novel GGSI, with a statin (lovastatin or pravastatin) in CD-1 mice. Twice-weekly dosing with VSW1198 at the previously established maximally tolerated dose in combination with a statin led to hepatotoxicity, while once-weekly VSW1198-based combinations were feasible. No abnormalities in kidney, spleen, brain or skeletal muscle were observed with combination therapy. Combination therapy disrupted protein geranylgeranylation in vivo. Evaluation of hepatic isoprenoid levels revealed decreased GGPP levels in the single drug groups and undetectable GGPP levels in the combination groups. Additional studies with combinations using 50% dose-reductions of either VSW1198 or lovastatin revealed minimal hepatotoxicity with expected on-target effects of diminished GGPP levels and disruption of protein geranylgeranylation. Combination statin/GGSI therapy significantly slowed tumor growth in a myeloma xenograft model. Collectively, these studies are the first to demonstrate that combination IBP inhibitor therapy alters isoprenoid levels and disrupts protein geranylgeranylation in vivo as well as slows tumor growth in a myeloma xenograft model, thus providing the framework for future clinical exploration.
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Vías Biosintéticas/efectos de los fármacos , Diterpenos/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Prenilación de Proteína/efectos de los fármacos , Terpenos/metabolismo , Triazoles/administración & dosificación , Animales , Vías Biosintéticas/fisiología , Línea Celular Tumoral , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Diterpenos/toxicidad , Evaluación Preclínica de Medicamentos/métodos , Quimioterapia Combinada , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/toxicidad , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/metabolismo , Femenino , Inhibidores de Hidroximetilglutaril-CoA Reductasas/toxicidad , Lovastatina/administración & dosificación , Lovastatina/toxicidad , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pravastatina/administración & dosificación , Pravastatina/toxicidad , Prenilación de Proteína/fisiología , Terpenos/antagonistas & inhibidores , Triazoles/toxicidad , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Zyxin is a member of the focal adhesion complex and plays a critical role in actin filament polymerization and cell motility. Several recent studies showed that Zyxin is a positive regulator of Yki/YAP (Yes-associated protein) signaling. However, little is known about the mechanisms by which Zyxin itself is regulated and how Zyxin affects Hippo-YAP activity. We first showed that Zyxin is phosphorylated by CDK1 during mitosis. Depletion of Zyxin resulted in significantly impaired colon cancer cell proliferation, migration, anchorage-independent growth, and tumor formation in xenograft animal models. Mitotic phosphorylation is required for Zyxin activity in promoting growth. Zyxin regulates YAP activity through the colon cancer oncogene CDK8. CDK8 knockout phenocopied Zyxin knockdown in colon cancer cells, while ectopic expression of CDK8 substantially restored the tumorigenic defects of Zyxin-depletion cells. Mechanistically, we showed that CDK8 directly phosphorylated YAP and promoted its activation. Fully activated YAP is required to support the growth in CDK8-knockout colon cancer cells in vitro and in vivo. Together, these observations suggest that Zyxin promotes colon cancer tumorigenesis in a mitotic-phosphorylation-dependent manner and through CDK8-mediated YAP activation.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Transformación Celular Neoplásica/metabolismo , Neoplasias del Colon/metabolismo , Quinasa 8 Dependiente de Ciclina/metabolismo , Mitosis , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Zixina/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Quinasa 8 Dependiente de Ciclina/genética , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Desnudos , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Fosforilación/genética , Factores de Transcripción , Proteínas Señalizadoras YAP , Zixina/genéticaRESUMEN
Enhanced free fatty acid (FFA) flux from adipose tissue (AT) to liver plays an important role in the development of nonalcoholic steatohepatitis (NASH) and alcohol-associated liver disease (AALD). We determined the effectiveness of nanoformulated superoxide dismutase 1 (Nano) in attenuating liver injury in a mouse model exhibiting a combination of NASH and AALD. Male C57BL6/J mice were fed a chow diet (CD) or a high-fat diet (HF) for 10 wk followed by pair feeding of the Lieber-DeCarli control (control) or ethanol (ET) diet for 4 wk. Nano was administered once every other day for the last 2 wk of ET feeding. Mice were divided into 1) CD + control diet (CD + Cont), 2) high-fat diet (HF) + control diet (HF + Cont), 3) HF + Cont + Nano, 4) HF + ET diet (HF + ET), and 5) HF + ET + Nano. The total fat mass, visceral AT mass (VAT), and VAT perilipin 1 content were significantly lower only in HF + ET-fed mice but not in HF + ET + Nano-treated mice compared with controls. The HF + ET-fed mice showed an upregulation of VAT CYP2E1 protein, and Nano abrogated this effect. We noted a significant rise in plasma FFAs, ALT, and monocyte chemoattractant protein-1 in HF + ET-fed mice, which was blunted in HF + ET + Nano-treated mice. HF + ET-induced increases in hepatic steatosis and inflammatory markers were attenuated upon Nano treatment. Nano reduced hepatic CYP2E1 and enhanced catalase levels in HF + ET-fed mice with a concomitant increase in SOD1 protein and activity in liver. Nano was effective in attenuating AT and liver injury in mice exhibiting a combination of NASH and AALD, partly via reduced CYP2E1-mediated ET metabolism in these organs.NEW & NOTEWORTHY Increased free fatty acid flux from adipose tissue (AT) to liver accompanied by oxidative stress promotes nonalcoholic steatohepatitis (NASH) and alcohol-associated liver injury (AALD). Obesity increases the severity of AALD. Using a two-hit model involving a high-fat diet and chronic ethanol feeding to mice, and treating them with nanoformulated superoxide dismutase (nanoSOD), we have shown that nanoSOD improves AT lipid storage, reduces CYP2E1 in AT and liver, and attenuates the combined NASH/AALD in mice.
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Citocromo P-450 CYP2E1/metabolismo , Hígado Graso Alcohólico/prevención & control , Grasa Intraabdominal/efectos de los fármacos , Hígado/efectos de los fármacos , Nanopartículas , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Superóxido Dismutasa-1/administración & dosificación , Adiposidad/efectos de los fármacos , Animales , Catalasa/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Modelos Animales de Enfermedad , Composición de Medicamentos , Hígado Graso Alcohólico/enzimología , Hígado Graso Alcohólico/genética , Hígado Graso Alcohólico/patología , Regulación de la Expresión Génica , Grasa Intraabdominal/enzimología , Grasa Intraabdominal/patología , Lipólisis/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Nanomedicina , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Estrés Oxidativo/efectos de los fármacos , Perilipina-1/genética , Perilipina-1/metabolismo , Transducción de Señal , Superóxido Dismutasa-1/químicaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a disease marked by high rates of mortality; it is mostly incurable at the time of diagnosis. Only about 7% of patients survive 5 years after diagnosis. Diagnosis at a late stage and rapid progression with minimal response to available treatments are the main reasons for this poor outcome. It is crucial to identify individuals at high risk of developing PDAC so preventive and early detection measures can be employed. Approximately 10% to 15% of PDAC cases have a hereditary or familial basis. In the majority of PDAC cases, no main causative gene has been identified, but several known germline pathogenic mutations have been shown to be related to an increased risk of this cancer. The presence of 2 or more patients with pancreatic cancer within the circle of first-degree relatives, without the presence of a causative germline mutation, is defined as familial pancreatic cancer; this accounts for 4% to 10% of PDAC. Based on the growing evidence supporting the benefit of germline genetic testing in patients with PDAC, both the American Society of Clinical Oncology and the National Comprehensive Cancer Network recently updated their guidelines to include recommendations around genetic testing for patients with pancreatic cancer. However, there is no general consensus on the group of patients and individuals who should be studied and screened. We present a demonstrative case and review the available data on hereditary and familial PDAC.
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Carcinoma Ductal Pancreático/genética , Carcinoma/genética , Mutación de Línea Germinal , Neoplasias Pancreáticas/genética , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma/diagnóstico por imagen , Carcinoma/terapia , Carcinoma Ductal Pancreático/diagnóstico por imagen , Carcinoma Ductal Pancreático/terapia , Pruebas Genéticas/métodos , Humanos , Masculino , Síndromes Neoplásicos Hereditarios/diagnóstico por imagen , Síndromes Neoplásicos Hereditarios/genética , Síndromes Neoplásicos Hereditarios/patología , Pancreatectomía/métodos , Neoplasias Pancreáticas/diagnóstico por imagen , Neoplasias Pancreáticas/terapia , Factores de RiesgoRESUMEN
Gastrointestinal stromal tumors (GISTs) are rare neoplasms of the gastrointestinal tract. They commonly present with nonspecific symptoms and thus are often discovered incidentally. They are best identified by CT scan, and most stain positive for CD117 (C-Kit), CD34, and/or DOG-1. Several risk stratification classification systems have been developed based on tumor size, mitotic rate, location, and perforation. Traditional chemotherapy and radiation therapy have been very ineffective, making surgery the mainstay of treatment. The discovery of mutations associated with these tumors has revolutionized the treatment approach. Imatinib mesylate, a selective tyrosine kinase receptor inhibitor, used as adjuvant or neoadjuvant therapy, has greatly improved the morbidity and mortality associated with GISTs. As the survival of patients has increased with the long-term use of targeted therapies, quality-of-life issues now have become much more relevant and have come to the forefront of care. We present a young woman who was successfully treated for GIST but now faces associated long-term adverse effects of imatinib, including the challenge of preserving fertility and the potential for childbearing.
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Preservación de la Fertilidad/métodos , Neoplasias Gastrointestinales/terapia , Tumores del Estroma Gastrointestinal/terapia , Mesilato de Imatinib/uso terapéutico , Adulto , Terapia Combinada , Femenino , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/patología , Neoplasias Gastrointestinales/cirugía , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/patología , Tumores del Estroma Gastrointestinal/cirugía , Humanos , Estadificación de Neoplasias , Inhibidores de Proteínas Quinasas/uso terapéutico , Resultado del TratamientoRESUMEN
BACKGROUND & AIMS: Cigarette smoking is a major risk factor for pancreatic cancer. Aggressive pancreatic tumors contain cancer cells with stem cell features. We investigated whether cigarette smoke induces stem cell features in pancreatic cancer cells. METHODS: KrasG12D; Pdx1-Cre mice were exposed to cigarette smoke or clean air (controls) for up to 20 weeks; pancreata were collected and analyzed by histology, quantitative reverse transcription polymerase chain reaction, and confocal immunofluorescence microscopy. HPNE and Capan1 cells were exposed to cigarette smoke extract (CSE), nicotine and nicotine-derived carcinogens (NNN or NNK), or clean air (controls) for 80 days and evaluated for stem cell markers and features using flow cytometry-based autofluorescence, sphere formation, and immunoblot assays. Proteins were knocked down in cells with small interfering RNAs. We performed RNA sequencing analyses of CSE-exposed cells. We used chromatin immunoprecipitation assays to confirm the binding of FOS-like 1, AP-1 transcription factor subunit (FOSL1) to RNA polymerase II-associated factor (PAF1) promoter. We obtained pancreatic ductal adenocarcinoma (PDAC) and matched nontumor tissues (n = 15) and performed immunohistochemical analyses. RESULTS: Chronic exposure of HPNE and Capan1 cells to CSE caused them to increase markers of stem cells, including autofluorescence and sphere formation, compared with control cells. These cells increased expression of ABCG2, SOX9, and PAF1, via cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) signaling to mitogen-activated protein kinase 1 and FOSL1. CSE-exposed pancreatic cells with knockdown of PAF1 did not show stem cell features. Exposure of cells to NNN and NNK led to increased expression of CHRNA7, FOSL1, and PAF1 along with stem cell features. Pancreata from KrasG12D; Pdx1-Cre mice exposed to cigarette smoke had increased levels of PAF1 mRNA and protein, compared with control mice, as well as increased expression of SOX9. Levels of PAF1 and FOSL1 were increased in PDAC tissues, especially those from smokers, compared with nontumor pancreatic tissue. CSE exposure increased expression of PHD-finger protein 5A, a pluripotent transcription factor and its interaction with PAF1. CONCLUSIONS: Exposure to cigarette smoke activates stem cell features of pancreatic cells, via CHRNA7 signaling and FOSL1 activation of PAF1 expression. Levels of PAF1 are increased in pancreatic tumors of humans and mice with chronic cigarette smoke exposure.
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Carcinoma Ductal Pancreático/metabolismo , Proteínas Portadoras/metabolismo , Fumar Cigarrillos/efectos adversos , Células Madre Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Animales , Carcinoma Ductal Pancreático/etiología , Línea Celular Tumoral , Humanos , Ratones , Páncreas/citología , Neoplasias Pancreáticas/etiología , Proteínas Proto-Oncogénicas c-fos/fisiología , Transducción de Señal/fisiología , Receptor Nicotínico de Acetilcolina alfa 7/fisiologíaRESUMEN
BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-ß-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. METHODS: We crossed C1galt1 floxed mice (C1galt1loxP/loxP) with KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n = 36) and poorly differentiated (n = 23) PDAC samples by immunohistochemistry. RESULTS: KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. CONCLUSIONS: In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.
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Adenocarcinoma/etiología , Galactosiltransferasas/fisiología , Neoplasias Pancreáticas/etiología , Adenocarcinoma/secundario , Animales , Sistemas CRISPR-Cas , Carcinoma Ductal Pancreático , Proliferación Celular , Galactosiltransferasas/genética , Glicosilación , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/patologíaRESUMEN
The enzyme geranylgeranyl diphosphate synthase (GGDPS) synthesizes the 20-carbon isoprenoid geranylgeranyl pyrophosphate, which is used in geranylgeranylation reactions. We have demonstrated that GGDPS inhibitors in multiple myeloma (MM) cells disrupt Rab geranylgeranylation, leading to inhibition of monoclonal protein trafficking, induction of the unfolded protein response pathway (UPR), and apoptosis. We have previously reported preclinical studies with the GGDPS inhibitor VSW1198, which is a mixture of homogeranyl/homoneryl triazole bisphosphonates. Additional structure-function efforts have led to development of the α-methylated derivatives RAM2093 (homogeranyl) and RAM2061 (homoneryl). As little is known regarding the impact of olefin stereochemistry on drug properties in vivo, we pursued additional preclinical evaluation of RAM2093 and RAM2061. In MM cell lines, both isomers induce activation of UPR/apoptotic markers in a concentration-dependent manner and with similar potency. Single-dose testing in CD-1 mice identified a maximum tolerated i.v. dose of 0.5 mg/kg for RAM2061 and 0.3 mg/kg for RAM2093. Liver toxicity was the primary barrier to dose escalation for both compounds. Disruption of geranylgeranylation in vivo was confirmed after multidose administration of either compound. Pharmacokinetic studies revealed plasma terminal half-lives of 29.2 ± 6 (RAM2061) and 22.1 ± 4 hours (RAM2093). Relative to RAM2061, RAM2093 levels were significantly higher in liver tissue but not in other tissues. Using MM.1S flank xenografts, we observed a significant reduction in tumor growth in mice treated with RAM2061 relative to controls. Collectively, these studies reveal olefin stereochemistry-dependent effects on GGDPS inhibitor biodistribution and confirm the in vivo efficacy of this novel therapeutic approach. SIGNIFICANCE STATEMENT: These studies reveal olefin stereochemistry-dependent effects on the in vivo properties of two novel triazole bisphosphonate inhibitors of geranylgeranyl diphosphate synthase and demonstrate the therapeutic potential of this class of inhibitors for the treatment of multiple myeloma.
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Alquenos/farmacología , Difosfonatos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Terpenos/farmacología , Distribución Tisular/efectos de los fármacos , Triazoles/farmacología , Alquenos/química , Alquenos/metabolismo , Animales , Difosfonatos/química , Difosfonatos/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Farnesiltransferasa/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Estereoisomerismo , Terpenos/química , Terpenos/metabolismo , Distribución Tisular/fisiología , Triazoles/química , Triazoles/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Geranylgeranyl diphosphate synthase (GGDPS) is the enzyme in the isoprenoid biosynthesis pathway that catalyzes the synthesis of the 20-carbon isoprenoid GGPP, which serves as the isoprenoid donor for protein geranylgeranylation reactions. Rab proteins mediate vesicle trafficking within the cell and their activity is dependent on geranylgeranylation. Our prior work has demonstrated that agents that disrupt Rab geranylgeranylation disrupt monoclonal protein trafficking in myeloma cells, resulting in induction of the unfolded protein response pathway and apoptosis. VSW1198 is a potent GGDPS inhibitor with measurable cellular activity at concentrations as low as 30 nM. Due to its potent activity against myeloma cells in vitro, we were interested in evaluating the toxicology profile, pharmacokinetic (PK) profile, tissue distribution pattern and metabolic stability of VSW1198 in preparation for in vivo efficacy studies. Single dose testing via IV administration in CD-1 mice revealed a maximum tolerated dose of 0.5 mg/kg. Doses ≥1 mg/kg resulted in liver toxicity that peaked around 6-7 days post-injection. Disruption of protein geranylgeranylation following repeat dosing of VSW1198 was confirmed via immunoblot analysis of unmodified Rap1a in multiple organs. The PK studies revealed a half-life of 47.7 ± 7.4 h. VSW1198 was present in all tested tissues with the highest levels in the liver. In both human liver microsomes and mouse S9 studies VSW1198 showed complete stability, suggesting no phase I or phase II metabolism. In summary, these studies demonstrate systemic distribution, on-target disruption of protein geranylgeranylation, and metabolic stability of a potent GGDPS inhibitor VSW1198 and form the basis for future efficacy studies in mouse models of myeloma.
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Antineoplásicos/farmacología , Farnesiltransferasa/antagonistas & inhibidores , Animales , Femenino , Humanos , Hígado/efectos de los fármacos , Dosis Máxima Tolerada , Ratones , Microsomas Hepáticos/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Prenilación de Proteína , Distribución TisularRESUMEN
Drug resistance is one of the main causes of colon cancer recurrence. However, our understanding of the underlying mechanisms and availability of therapeutic options remains limited. Here we show that expression of pyruvate dehydrogenase kinase 4 (PDK4) is positively correlated with drug resistance of colon cancer cells and induced by 5-fluorouracil (5-FU) treatment in drug-resistant but not drug-sensitive cells. Knockdown of PDK4 expression sensitizes colon cancer cells to 5-FU or oxaliplatin-induced apoptosis in vitro and increases the effectiveness of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo In addition, we demonstrate for the first time that TGFß mediates drug resistance by regulating PDK4 expression and that 5-FU induces PDK4 expression in a TGFß signaling-dependent manner. Mechanistically, knockdown or inhibition of PDK4 significantly increases the inhibitory effect of 5-FU on expression of the anti-apoptotic factors Bcl-2 and survivin. Importantly, studies of patient samples indicate that expression of PDK4 and phosphorylation of Smad2, an indicator of TGFß pathway activation, show a strong correlation and that both positively associate with chemoresistance in colorectal cancer. These findings indicate that the TGFß/PDK4 signaling axis plays an important role in the response of colorectal cancer to chemotherapy. A major implication of our studies is that inhibition of PDK4 may have considerable therapeutic potential to overcome drug resistance in colorectal cancer patients, which warrants the development of PDK4-specific inhibitors.
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Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Proteínas Serina-Treonina Quinasas/biosíntesis , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Fluorouracilo/farmacología , Humanos , Ratones , Ratones Desnudos , Compuestos Organoplatinos/farmacología , Oxaliplatino , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Proteína Smad2/biosíntesis , Proteína Smad2/genética , Factor de Crecimiento Transformador beta/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Objective: To characterize the expression of malondialdehdye-acetaldehyde (MAA) adducts and anti-MAA antibody in articular tissues and serum of patients with RA. Methods: Paired sera and SF were examined from 29 RA and 13 OA patients. Anti-MAA antibody, RF, ACPA and total immunoglobulin were quantified. SF-serum measures were compared within and between disease groups. The presence and co-localization of MAA, citrulline and select leukocyte antigens in RA and OA synovial tissues were examined using immunohistochemistry. Results: Circulating and SF anti-MAA antibody concentrations were higher in RA vs OA by 1.5- to 5-fold. IgG (P < 0.001), IgM (P = 0.006) and IgA (P = 0.036) anti-MAA antibodies were higher in paired RA SF than serum, differences not observed for total immunoglobulin, RF or ACPA. In RA synovial tissues, co-localization of MAA with citrulline and CD19+ or CD27+ B cells was demonstrated and was much higher in magnitude than MAA or citrulline co-localization with T cells, monocytes, macrophages or dendritic cells (P < 0.01). Conclusion: Anti-MAA antibodies are present in higher concentrations in the RA joint compared with sera, a finding not observed for other disease-related autoantibodies. Co-localization of MAA and citrulline with mature B cells, coupled with the local enrichment of anti-MAA immune responses, implicates MAA-adduct formation in local autoantibody production.
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Acetaldehído/inmunología , Artritis Reumatoide/inmunología , Autoanticuerpos/análisis , Articulaciones/inmunología , Malondialdehído/inmunología , Anciano , Artritis Reumatoide/sangre , Estudios de Casos y Controles , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Osteoartritis/sangre , Osteoartritis/inmunología , Factor Reumatoide/sangre , Líquido Sinovial/inmunologíaRESUMEN
Development of drug resistance is one of the major causes of colorectal cancer recurrence, yet mechanistic understanding and therapeutic options remain limited. Here, we show that expression of microRNA (miR)-520g is correlated with drug resistance of colon cancer cells. Ectopic expression of miR-520g conferred resistance to 5-fluorouracil (5-FU)- or oxaliplatin-induced apoptosis in vitro and reduced the effectiveness of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo. Further studies indicated that miR-520g mediated drug resistance through down-regulation of p21 expression. Moreover, p53 suppressed miR-520g expression, and deletion of p53 up-regulated miR-520g expression. Inhibition of miR-520g in p53(-/-) cells increased their sensitivity to 5-FU treatment. Importantly, studies of patient samples indicated that expression of miR-520g correlated with chemoresistance in colorectal cancer. These findings indicate that the p53/miR-520g/p21 signaling axis plays an important role in the response of colorectal cancer to chemotherapy. A major implication of our studies is that inhibition of miR-520g or restoration of p21 expression may have considerable therapeutic potential to overcome drug resistance in colorectal cancer patients, especially in those with mutant p53.
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Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Resistencia a Antineoplásicos/genética , MicroARNs/genética , Animales , Neoplasias Colorrectales/patología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Fluorouracilo/uso terapéutico , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Ratones , MicroARNs/biosíntesis , Mutación , Compuestos Organoplatinos/uso terapéutico , Oxaliplatino , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: Hepatic dysfunction is a recognized complication after Fontan palliation of congenital heart disease. We sought to quantitatively measure hepatic stiffness and vascular Doppler indices using ultrasound (US) and shear wave elastography (SWE) in a Fontan cohort. Subjects were prospectively recruited for echocardiography and real-time hepatic duplex US with SWE for hepatic stiffness (kPa). Doppler peak velocities, velocity time integral, resistive, pulsatility, acceleration indices (RI, PI, AI), and flow volume were measured in celiac artery, superior mesenteric artery, and main portal vein (MPV). A subset underwent cardiac catheterizations with liver biopsy. Correlations were explored between SWE, duplex, hemodynamic, and histopathologic data. In all, 106 subjects were studied including 41 patients with Fontan physiology (age 13.8 ± 6 years, weight 45.4 ± 23 kg) and 65 controls (age 15.0 ± 8.4 years, weight 47.9 ± 22 kg). Patients with Fontan physiology had significantly higher hepatic stiffness (15.6 versus 5.5 kPa, P < 0.0001), higher celiac RI (0.78 versus 0.73, P = 0.04) superior mesenteric artery RI (0.89 versus 0.84, P = 0.005), and celiac PI (1.87 versus 1.6, P = 0.034); while MPV flow volume (287 versus 420 mL/min in controls, P = 0.007) and SMA AI (829 versus 1100, P = 0.002) were lower. Significant correlation was seen for stiffness with ventricular end-diastolic pressure (P = 0.001) and pulmonary artery wedge pressure (P = 0.009). Greater stiffness correlated with greater degrees of histopathologic fibrosis. No significant change was seen in stiffness or other duplex indices with age, gender, time since Fontan, or ventricular morphology. CONCLUSION: Elevated hepatic afterload in Fontan, manifested by high ventricular end-diastolic pressures and pulmonary arterial wedge pressures, is associated with remarkably increased hepatic stiffness, abnormal vascular flow patterns, and fibrotic histologic changes. The MPV is dilated and carries decreased flow volume, while the celiac and superior mesenteric arterial RI is increased. SWE is feasible in this population and shows promise as a means for predicting disease severity on liver biopsy.
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Procedimiento de Fontan/efectos adversos , Cirrosis Hepática/etiología , Adolescente , Adulto , Cateterismo Cardíaco , Estudios de Casos y Controles , Niño , Preescolar , Ecocardiografía , Diagnóstico por Imagen de Elasticidad , Femenino , Procedimiento de Fontan/estadística & datos numéricos , Voluntarios Sanos , Humanos , Hígado/diagnóstico por imagen , Hígado/patología , Cirrosis Hepática/diagnóstico por imagen , Cirrosis Hepática/patología , Masculino , Estudios Prospectivos , Ultrasonografía Doppler Dúplex , Adulto JovenRESUMEN
GVHD has been reported in 8-10% of children after small bowel transplant (SBTx). Immunodeficient children may be predisposed to aggressive, steroid-resistant GVHD. There exists a unique association of immunodeficiency in children with MIA (MIAI). We report on our SBTx experience in patients with the diagnosis of MIAI, their high incidence of GVHD, and the possible role of stem cell transplantation in these patients. We performed a review of records from children that underwent SBTx or that we evaluated for SBTx at our institution. We focused on the diagnoses of atresia, multiple intestinal atresia, immunodeficiency, and GVHD in our patient population. Children with MIAI are likely to experience severe GVHD following SBTx. MIAI correlated with a 100% incidence of GVHD in these patients. Of the five patients with MIAI that underwent SBTx, three succumbed to severe GVHD within 1-6 months after SBTx. One patient received stem cell transplant prior to SBTx and did not develop severe GVHD, but died from influenza nine months after SBTx. Our unique patient survives long-term, with engraftment of donor γ δ T cells. He has mild, persistent chronic GVHD. Atresia is a common referral diagnosis for SBTx. Patients with multiple atresias, especially MIAI, are at significant risk for the complication of GVHD following SBTx. We recommend careful immunologic assessment and antecedent stem cell transplant in children with MIAI prior to SBTx.
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Síndromes de Inmunodeficiencia/cirugía , Atresia Intestinal/cirugía , Intestinos/trasplante , Adolescente , Niño , Preescolar , Femenino , Enfermedad Injerto contra Huésped/prevención & control , Enfermedad Injerto contra Huésped/terapia , Humanos , Lactante , Estimación de Kaplan-Meier , Masculino , Estudios Retrospectivos , Trasplante de Células Madre , Linfocitos T/metabolismo , Resultado del TratamientoRESUMEN
Nasopharyngeal carcinoma is an endemic entity with a strong association with Epstein-Barr virus and a new recognition of human papilloma virus-mediated effects in nonendemic areas. Here, we discuss a nasopharyngeal carcinoma suspected as based on imaging results with metastasis to the lymph nodes, lung, spleen, bone, and liver. Gross and microscopic findings from the autopsy were clinicopathologically correlated with antemortem clinical studies and investigations. The authors report a case of EBV- and HPV-negative nonendemic, multisite metastatic nasopharyngeal carcinoma, shown to be nonkeratinizing undifferentiated subtype.