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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: 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: 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
Glioblastoma (GBM) is amongst the most aggressive brain tumors with a dismal prognosis. Despite significant advances in the current multimodality therapy including surgery, postoperative radiotherapy (RT) and temozolomide (TMZ)-based concomitant and adjuvant chemotherapy (CT), tumor recurrence is nearly universal with poor patient outcomes. These limitations are in part due to poor drug penetration through the blood-brain barrier (BBB) and resistance to CT and RT by a small population of cancer cells recognized as tumor-initiating cells or cancer stem cells (CSCs). Though CT and RT kill the bulk of the tumor cells, they fail to affect CSCs, resulting in their enrichment and their development into more refractory tumors. Therefore, identifying the mechanisms of resistance and developing therapies that specifically target CSCs can improve response, prevent the development of refractory tumors and increase overall survival of GBM patients. Small molecule inhibitors that can breach the BBB and selectively target CSCs are emerging. In this review, we have summarized the recent advancements in understanding the GBM CSC-specific signaling pathways, the CSC-tumor microenvironment niche that contributes to CT and RT resistance and the use of novel combination therapies of small molecule inhibitors that may be used in conjunction with TMZ-based chemoradiation for effective management of GBM.
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Barrera Hematoencefálica/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Encefálicas/radioterapia , Quimioradioterapia , Resistencia a Antineoplásicos , Glioblastoma/radioterapia , Humanos , Tolerancia a Radiación , Microambiente TumoralRESUMEN
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
Despite therapeutic advances, overall survival in glioblastoma is dismal. To optimize progress, a more detailed understanding of glioma's molecular, cellular, and intercellular pathophysiology is needed. Recent investigation has revealed a vital role for exosomes in inter-cellular signaling, tumor cell support, and regulation of the tumor microenvironment. Exosomes carry miRNAs, lncRNAs, mRNAs, proteins, immune regulatory molecules, nucleic acids, and lipids; however, the composition of exosome cargo is variable depending on the cell of origin. Specific exosomal miRNA contents such as miR-21, miR-301a, miR-151a, miR-148a, and miR-5096 are altered in high-grade glioma. Unique proteomic, genomic, and miRNA signatures of tumor exosomes have been associated with disease pathobiology, temozolomide resistance, immunosuppression, and tumor proliferation. Exosomes hold promise for tissue diagnostic glioma diagnosis and monitoring response to therapy. This review summarizes the current understanding of exosomes, their crucial role in glioma pathology, and future directions for their use in diagnosis and treatment. METHODS: The MEDLINE/PubMed database was reviewed for papers written in English and publication dates of 1981-2023, using the search string "Exosome", "Extracellular vesicles", "Glioma", "Exosomes in glioma".
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Exosomas , Glioblastoma , Glioma , MicroARNs , Humanos , Exosomas/metabolismo , Proteómica , Glioma/diagnóstico , Glioma/genética , Glioma/terapia , MicroARNs/genética , Glioblastoma/patología , Línea Celular Tumoral , Microambiente TumoralRESUMEN
Glioblastoma (GBM), an extremely aggressive and prevalent malignant brain tumor, remains a challenge to treat. Despite a multimodality treatment approach, GBM recurrence remains inevitable, particularly with the emergence of temozolomide (TMZ) resistance and limited treatment options. Surprisingly, previous studies show that a history of allergies, atopy, or asthma is inversely associated with GBM risk. Further, the electronic medical record at the University Hospital of Lausanne showed that the GBM patients taking antihistamine during treatment had better survival. Histamine is an essential neurotransmitter in the brain and plays a significant role in regulating sleep, hormonal balance, and cognitive functions. Elevated levels of histamine and increased histamine receptor expression have been found in different tumors and their microenvironments, including GBM. High histamine 1 receptor (HRH1) expression is inversely related to overall and progression-free survival in GBM patients, further emphasizing the role of histamine in disease progression. This review aims to provide insights into the challenges of GBM treatment, the role of histamine in GBM progression, and the rationale for considering antihistamines as targeted therapy. The review concludes by encouraging further investigation into antihistamine mechanisms and their impact on the tumor microenvironment.
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Diffuse intrinsic pontine glioma (DIPG) is a childhood malignancy of the brainstem with a dismal prognosis. Despite recent advances in its understanding at the molecular level, the prognosis of DIPG has remained unchanged. This article aims to review the current understanding of the genetic pathophysiology of DIPG and to highlight promising therapeutic targets. Various DIPG treatment strategies have been investigated in pre-clinical studies, several of which have shown promise and have been subsequently translated into ongoing clinical trials. Ultimately, a multifaceted therapeutic approach that targets cell-intrinsic alterations, the micro-environment, and augments the immune system will likely be necessary to eradicate DIPG.
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Neoplasias del Tronco Encefálico , Glioma Pontino Intrínseco Difuso , Humanos , Glioma Pontino Intrínseco Difuso/genética , Glioma Pontino Intrínseco Difuso/terapia , Glioma Pontino Intrínseco Difuso/patología , Glioma Pontino Intrínseco Difuso/tratamiento farmacológico , Neoplasias del Tronco Encefálico/genética , Neoplasias del Tronco Encefálico/terapia , Neoplasias del Tronco Encefálico/patología , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Pronóstico , Microambiente Tumoral , Terapia Molecular Dirigida/métodosRESUMEN
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
Aberrant protein glycosylation has been shown to have a significant contribution in aggressive cancer, including pancreatic cancer (PC). Emerging evidence has implicated the involvement of cancer stem cells (CSCs) in PC aggressiveness; however, the contribution of glycosylation on self-renewal properties and maintenance of CSC is understudied. Here, using several in vitro and in vivo models lacking C1GALT1 expression, we identified the role of aberrant O-glycosylation in stemness properties and aggressive PC metastasis. A loss in C1GALT1 was found to result in the truncation of O-glycosylation on several glycoproteins with an enrichment of Tn carbohydrate antigen. Mapping of Tn-bearing glycoproteins in C1GALT1 KO cells identified significant Tn enrichment on CSC glycoprotein CD44. Notably, a loss of C1GALT1 in PC cells was found to enhance CSC features (side population-SP, ALDH1+, and tumorspheres) and self-renewal markers NANOG, SOX9, and KLF4. Furthermore, a loss of CD44 in existing C1GALT1 KO cells decreased NANOG expression and CSC features. We determined that O-glycosylation of CD44 activates ERK/NF-kB signaling, which results in increased NANOG expression in PC cells that facilitated the alteration of CSC features, suggesting that NANOG is essential for PC stemness. Finally, we identified that loss of C1GALT1 expression was found to augment tumorigenic and metastatic potential, while an additional loss of CD44 in these cells reversed the effects. Overall, our results identified that truncation of O-glycans on CD44 increases NANOG activation that mediates increased CSC activation.
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Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/fisiología , Neoplasias Pancreáticas/genética , Diferenciación Celular , Línea Celular Tumoral , Glicosilación , Humanos , Metástasis de la Neoplasia , Células Madre Neoplásicas/patología , Neoplasias Pancreáticas/patologíaRESUMEN
Bone metastases occur in patients with advanced-stage prostate cancer (PCa). The cell-cell interaction between PCa and the bone microenvironment forms a vicious cycle that modulates the bone microenvironment, increases bone deformities, and drives tumor growth in the bone. However, the molecular mechanisms of PCa-mediated modulation of the bone microenvironment are complex and remain poorly defined. Here, we evaluated growth differentiation factor-15 (GDF15) function using in vivo preclinical PCa-bone metastasis mouse models and an in vitro bone cell coculture system. Our results suggest that PCa-secreted GDF15 promotes bone metastases and induces bone microarchitectural alterations in a preclinical xenograft model. Mechanistic studies revealed that GDF15 increases osteoblast function and facilitates the growth of PCa in bone by activating osteoclastogenesis through osteoblastic production of CCL2 and RANKL and recruitment of osteomacs. Altogether, our findings demonstrate the critical role of GDF15 in the modulation of the bone microenvironment and subsequent development of PCa bone metastasis.
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BACKGROUND: Radiation therapy (RT) has a suboptimal effect in patients with pancreatic ductal adenocarcinoma (PDAC) due to intrinsic and acquired radioresistance (RR). Comprehensive bioinformatics and microarray analysis revealed that cholesterol biosynthesis (CBS) is involved in the RR of PDAC. We now tested the inhibition of the CBS pathway enzyme, farnesyl diphosphate synthase (FDPS), by zoledronic acid (Zol) to enhance radiation and activate immune cells. METHODS: We investigated the role of FDPS in PDAC RR using the following methods: in vitro cell-based assay, immunohistochemistry, immunofluorescence, immunoblot, cell-based cholesterol assay, RNA sequencing, tumouroids (KPC-murine and PDAC patient-derived), orthotopic models, and PDAC patient's clinical study. FINDINGS: FDPS overexpression in PDAC tissues and cells (P < 0.01 and P < 0.05) is associated with poor RT response and survival (P = 0.024). CRISPR/Cas9 and pharmacological inhibition (Zol) of FDPS in human and mouse syngeneic PDAC cells in conjunction with RT conferred higher PDAC radiosensitivity in vitro (P < 0.05, P < 0.01, and P < 0.001) and in vivo (P < 0.05). Interestingly, murine (P = 0.01) and human (P = 0.0159) tumouroids treated with Zol+RT showed a significant growth reduction. Mechanistically, RNA-Seq analysis of the PDAC xenografts and patients-PBMCs revealed that Zol exerts radiosensitization by affecting Rac1 and Rho prenylation, thereby modulating DNA damage and radiation response signalling along with improved systemic immune cells activation. An ongoing phase I/II trial (NCT03073785) showed improved failure-free survival (FFS), enhanced immune cell activation, and decreased microenvironment-related genes upon Zol+RT treatment. INTERPRETATION: Our findings suggest that FDPS is a novel radiosensitization target for PDAC therapy. This study also provides a rationale to utilize Zol as a potential radiosensitizer and as an immunomodulator in PDAC and other cancers. FUNDING: National Institutes of Health (P50, P01, and R01).
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/radioterapia , Línea Celular Tumoral , Proliferación Celular , Daño del ADN , Regulación Neoplásica de la Expresión Génica , Geraniltranstransferasa/genética , Geraniltranstransferasa/metabolismo , Humanos , Ratones , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/radioterapia , Transducción de Señal , Microambiente Tumoral/genética , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Haploinsufficiency of chromosome 17p and c-Myc amplification distinguish group 3 medulloblastomas which are associated with early metastasis, rapid recurrence, and swift mortality. Tumor suppressor genes on this locus have not been adequately characterized. We elucidated the role of miR-212-3p in the pathophysiology of group 3 tumors. First, we learned that miR-212-3p undergoes epigenetic silencing by histone modifications in group 3 tumors. Restoring its expression reduced cancer cell proliferation, migration, colony formation, and wound healing in vitro and attenuated tumor burden and improved survival in vivo. MiR-212-3p also triggered c-Myc destabilization and degradation, leading to elevated apoptosis. We then isolated an oncogenic target of miR-212-3p, i.e. NFIB, a nuclear transcription factor implicated in metastasis and recurrence in various cancers. Increased expression of NFIB was confirmed in group 3 tumors and associated with poor survival. NFIB silencing reduced cancer cell proliferation, migration, and invasion. Concurrently, reduced medullosphere formation and stem cell markers (Nanog, Oct4, Sox2, CD133) were noted. These results substantiate the tumor-suppressive role of miR-212-3p in group 3 MB and identify a novel oncogenic target implicated in metastasis and tumor recurrence.
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Neoplasias Cerebelosas/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Meduloblastoma/metabolismo , MicroARNs/metabolismo , Factores de Transcripción NFI/metabolismo , Animales , Células Cultivadas , Neoplasias Cerebelosas/genética , Modelos Animales de Enfermedad , Humanos , Meduloblastoma/genética , Ratones , MicroARNs/genética , Factores de Transcripción NFI/genéticaRESUMEN
BACKGROUND: Glioblastoma (GBM) has a devastating median survival of only one year. Treatment includes resection, radiation therapy, and temozolomide (TMZ); however, the latter increased median survival by only 2.5 months in the pivotal study. A desperate need remains to find an effective treatment. METHODS: We used the Connectivity Map (CMap) bioinformatic tool to identify candidates for repurposing based on GBM's specific genetic profile. CMap identified histone deacetylase (HDAC) inhibitors as top candidates. In addition, Gene Expression Profiling Interactive Analysis (GEPIA) identified HDAC1 and HDAC2 as the most upregulated and HDAC11 as the most downregulated HDACs. We selected PCI-24781/abexinostat due to its specificity against HDAC1 and HDAC2, but not HDAC11, and blood-brain barrier permeability. RESULTS: We tested PCI-24781 using in vitro human and mouse GBM syngeneic cell lines, an in vivo murine orthograft, and a genetically engineered mouse model for GBM (PEPG - PTENflox/+; EGFRvIII+; p16Flox/- & GFAP Cre +). PCI-24781 significantly inhibited tumor growth and downregulated DNA repair machinery (BRCA1, CHK1, RAD51, and O6-methylguanine-DNA- methyltransferase (MGMT)), increasing DNA double-strand breaks and causing apoptosis in the GBM cell lines, including an MGMT expressing cell line in vitro. Further, PCI-24781 decreased tumor burden in a PEPG GBM mouse model. Notably, TMZ + PCI increased survival in orthotopic murine models compared to TMZ + vorinostat, a pan-HDAC inhibitor that proved unsuccessful in clinical trials. CONCLUSION: PCI-24781 is a novel GBM-signature specific HDAC inhibitor that works synergistically with TMZ to enhance TMZ efficacy and improve GBM survival. These promising MGMT-agnostic results warrant clinical evaluation.
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Antineoplásicos Alquilantes/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Temozolomida/farmacología , Animales , Antineoplásicos Alquilantes/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular , Biología Computacional/métodos , Roturas del ADN , Manejo de la Enfermedad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas/métodos , Resistencia a Antineoplásicos/genética , Perfilación de la Expresión Génica , Glioblastoma/diagnóstico , Glioblastoma/tratamiento farmacológico , Humanos , Ratones , Ratones Transgénicos , Temozolomida/uso terapéutico , Transcriptoma , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Metabolic reprogramming and cancer stem cells drive the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). However, the metabolic and stemness programs of pancreatic precursor lesions (PPL), considered early PDAC development events, have not been thoroughly explored. EXPERIMENTAL DESIGN: Meta-analyses using gene expression profile data from NCBI Gene Expression Omnibus and IHC on tissue microarrays (TMA) were performed. The following animal and cellular models were used: cerulean-induced KrasG12D; Pdx1 Cre (KC) acinar-to-ductal metaplasia (ADM) mice, KrasG12D; Smad4Loss; Pdx-1 Cre (KCSmad4-) intraductal papillary mucinous neoplasm (IPMN) mice, LGKC1 cell line derived from the doxycycline-inducible Gnas IPMN model, and human IPMN organoids. Flow cytometry, Seahorse extracellular flux analyzer, qRT-PCR, and sphere assay were used to analyze metabolic and stemness features. SR18292 was used to inhibit PGC1α, and short hairpin RNA was used to knockdown (KD) PGC1α. RESULTS: The meta-analysis revealed a significant upregulation of specific stemness genes in ADM-mediated pancreatic intraepithelial neoplasms (PanIN) and IPMN. Meta- and TMA analyses followed by in vitro and in vivo validation revealed that ADM/PanIN exhibit increased PGC1α and oxidative phosphorylation (OXPhos) but reduced CPT1A. IPMN showed elevated PGC1α, fatty acid ß-oxidation (FAO) gene expression, and FAO-OXPhos. PGC1α was co-overexpressed with its coactivator NRF1 in ADM/PanINs and with PPARγ in IPMN. PGC1α KD or SR18292 inhibited the specific metabolic and stemness features of PPLs and repressed IPMN organoid growth. CONCLUSIONS: ADM/PanINs and IPMNs show specific stemness signatures with unique metabolisms. Inhibition of PGC1α using SR18292 diminishes the specific stemness by targeting FAO-independent and FAO-dependent OXPhos of ADM/PanINs and IPMNs, respectively.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Carcinoma Ductal Pancreático/patología , Humanos , Ratones , Páncreas/patología , Conductos Pancreáticos/patología , Neoplasias Pancreáticas/patología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismoRESUMEN
Lung cancer (LC) is the leading cause of cancer-related mortality. However, the molecular mechanisms associated with the development of metastasis are poorly understood. Understanding the biology of LC metastasis is critical to unveil the molecular mechanisms for designing targeted therapies. We developed two genetically engineered LC mouse models KrasG12D/+ ; Trp53R172H/+ ; Ad-Cre (KPA) and KrasG12D/+ ; Ad-Cre (KA). Survival analysis showed significantly (P = 0.0049) shorter survival in KPA tumor-bearing mice as compared to KA, suggesting the aggressiveness of the model. Our transcriptomic data showed high expression of N-acetylgalactosaminide alpha-2, 6-sialyltransferase 1 (St6galnac-I) in KPA compared to KA tumors. ST6GalNAc-I is an O-glycosyltransferase, which catalyzes the addition of sialic acid to the initiating GalNAc residues forming sialyl Tn (STn) on glycoproteins, such as mucins. Ectopic expression of species-specific p53 mutants in the syngeneic mouse and human LC cells led to increased cell migration and high expression of ST6GalNAc-I, STn, and MUC5AC. Immunoprecipitation of MUC5AC in the ectopically expressing p53R175H cells exhibited higher affinity toward STn. In addition, ST6GalNAc-I knockout (KO) cells also showed decreased migration, possibly due to reduced glycosylation of MUC5AC as observed by low STn on the glycoprotein. Interestingly, ST6GalNAc-I KO cells injected mice developed less liver metastasis (P = 0.01) compared to controls, while colocalization of MUC5AC and STn was observed in the liver metastatic tissues of control mice. Collectively, our findings support the hypothesis that mutant p53R175H mediates ST6GalNAc-I expression, leading to the sialyation of MUC5AC, and thus contribute to LC liver metastasis.
Asunto(s)
Neoplasias Hepáticas , Neoplasias Pulmonares , Animales , Glicosilación , Humanos , Neoplasias Pulmonares/genética , Ratones , Mucina 5AC/metabolismo , Ácido N-Acetilneuramínico , Sialiltransferasas/genética , Sialiltransferasas/metabolismoRESUMEN
Pancreatic cancer (PC) is difficult to defeat due to mechanism (s) driving metastasis and drug resistance. Cancer stemness is a major challenging phenomenon associated with PC metastasis and limiting therapy efficacy. In this study, we evaluated the pre-clinical and clinical significance of eradicating pancreatic cancer stem cells (PCSC) and its components using a pan-EGFR inhibitor afatinib in combination with gemcitabine. Afatinib in combination with gemcitabine significantly reduced KrasG12D/+; Pdx-1 Cre (KC) (P < 0.01) and KrasG12D/+; p53R172H/+; Pdx-1 Cre (KPC) (P < 0.05) derived mouse tumoroids and KPC-derived murine syngeneic cell line growth compared to gemcitabine/afatinib alone treatment. The drug combination also reduced PC xenograft tumor burden (P < 0.05) and the incidence of metastasis by affecting key stemness markers, as confirmed by co-localization studies. Moreover, the drug combination significantly decreases the growth of various PC patient-derived organoids (P < 0.001). We found that SOX9 is significantly overexpressed in high-grade PC tumors (P < 0.05) and in chemotherapy-treated patients compared to chemo-naïve patients (P < 0.05). These results were further validated using publicly available datasets. Moreover, afatinib alone or in combination with gemcitabine decreased stemness and tumorspheres by reducing phosphorylation of EGFR family proteins, ERK, FAK, and CSC markers. Mechanistically, afatinib treatment decreased CSC markers by downregulating SOX9 via FOXA2. Indeed, EGFR and FOXA2 depletion reduced SOX9 expression in PCSCs. Taken together, pan-EGFR inhibition by afatinib impedes PCSCs growth and metastasis via the EGFR/ERK/FOXA2/SOX9 axis. This novel mechanism of pan-EGFR inhibitor and its ability to eradicate CSC may serve as a tailor-made approach to enhance chemotherapeutic benefits in other cancer types.
Asunto(s)
Factor Nuclear 3-beta del Hepatocito/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Factor de Transcripción SOX9/antagonistas & inhibidores , Afatinib/uso terapéutico , Animales , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/fisiología , Factor Nuclear 3-beta del Hepatocito/fisiología , Humanos , Ratones , Metástasis de la Neoplasia , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Pancreáticas/patología , Factor de Transcripción SOX9/fisiología , GemcitabinaRESUMEN
Pancreatic cancer (PC) is acquired postnatally; to mimic this scenario, we developed an inducible KrasG12D; Ptf1a-CreER™ (iKC) mouse model, in which Kras is activated postnatally at week 16 upon tamoxifen (TAM) administration. Upon TAM treatment, iKC mice develop pancreatic intraepithelial neoplasia (PanIN) lesions and PC with metastasis at the fourth and fortieth weeks, respectively, and exhibited acinar-to-ductal metaplasia (ADM) and transdifferentiation. Kras activation upregulated the transcription factors Ncoa3, p-cJun and FoxM1, which in turn upregulated expression of transmembrane mucins (Muc1, Muc4 and Muc16) and secretory mucin (Muc5Ac). Interestingly, knockdown of KrasG12D in multiple PC cell lines resulted in downregulation of MUC1, MUC4, MUC5AC and MUC16. In addition, iKC mice exhibited ADM and transdifferentiation. Our results show that the iKC mouse more closely mimics human PC development and can be used to investigate pancreatic ductal adenocarcinoma (PDAC) biomarkers, early onset of PDAC, and ADM. The iKC model can also be used for preclinical strategies such as targeting mucin axis alone or in combination with neo-adjuvant, immunotherapeutic approaches and to monitor chemotherapy response.
Asunto(s)
Carcinoma Ductal Pancreático/etiología , Carcinoma Ductal Pancreático/patología , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/genética , Mucinas/genética , Neoplasias Pancreáticas/etiología , Neoplasias Pancreáticas/patología , Tamoxifeno/efectos adversos , Animales , Biomarcadores , Carcinoma Ductal Pancreático/metabolismo , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Expresión Génica , Inmunohistoquímica , Ratones , Mucinas/metabolismo , Familia de Multigenes , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Factores de TranscripciónRESUMEN
PURPOSE: Docetaxel plays an indispensable role in the management of advanced prostate cancer. However, more than half of patients do not respond to docetaxel, and those good responders frequently experience significant cumulative toxicity, which limits its dose duration and intensity. Hence, a second agent that could increase the initial efficacy of docetaxel and maintain tolerability at biologically effective doses may improve outcomes for patients. EXPERIMENTAL DESIGN: We determined phosphodiesterase 5 (PDE5) expression levels in human and genetically engineered mouse (GEM) prostate tissues and tumor-derived cell lines. Furthermore, we investigated the therapeutic benefits and underlying mechanism of PDE5 inhibitor sildenafil in combination with docetaxel using in vitro, Pten conditional knockout (cKO), derived tumoroid and xenograft prostate cancer models. RESULTS: PDE5 expression was higher in both human and mouse prostate tumors and cancer cell lines compared with normal tissues/cells. In GEM prostate-derived cell lines, PDE5 expression increased from normal prostate (wild-type) epithelial cells to androgen-dependent and castrated prostate-derived cell lines. The addition of physiologically achievable concentrations of sildenafil enhanced docetaxel-induced prostate cancer cell growth inhibition and apoptosis in vitro, reduced murine 3D tumoroid growth, and in vivo tumorigenicity as compared with docetaxel alone. Furthermore, sildenafil enhanced docetaxel-induced NO and cGMP levels thereby augmenting antitumor activity. CONCLUSIONS: Our results demonstrate that sildenafil's addition could sensitize docetaxel chemotherapy in prostate cancer cells at much lesser concentration than needed for inducing cell death. Thus, the combinatorial treatment of sildenafil and docetaxel may improve anticancer efficacy and reduce chemotherapy-induced side-effects among patients with advanced prostate cancer.
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Docetaxel/administración & dosificación , Óxido Nítrico/genética , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Citrato de Sildenafil/administración & dosificación , Andrógenos/genética , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , AMP Cíclico/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/genética , Modelos Animales de Enfermedad , Docetaxel/efectos adversos , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Noqueados , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Transducción de Señal/efectos de los fármacos , Citrato de Sildenafil/efectos adversosRESUMEN
BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor with universal recurrence and poor prognosis. The recurrence is largely driven by chemoradiation resistant cancer stem cells (CSCs). Epidermal growth factor receptor (EGFR) and its mutant EGFRvIII are amplified in ~ 60% and ~ 30% of GBM patients, respectively; however, therapies targeting EGFR have failed to improve disease outcome. EGFRvIII-mediated cross-activation of tyrosine kinase receptor, cMET, regulates GBM CSC maintenance and promote tumor recurrence. Here, we evaluated the efficacy of pan-EGFR inhibitor afatinib and Temozolomide (TMZ) combination on GBM in vitro and in vivo. METHODS: We analyzed the effect of afatinib and temozolomide (TMZ) combination on GBM cells U87MG and U251 engineered to express wild type (WT) EGFR, EGFRvIII or EGFRvIII dead kinase, CSCs isolated from U87 and U87EGFRvIII in vitro. The therapeutic utility of the drug combination was investigated on tumor growth and progression using intracranially injected U87EGFRvIII GBM xenografts. RESULTS: Afatinib and TMZ combination synergistically inhibited the proliferation, clonogenic survival, motility, invasion and induced senescence of GBM cells compared to monotherapy. Mechanistically, afatinib decreased U87EGFRvIII GBM cell proliferation and motility/invasion by inhibiting EGFRvIII/AKT, EGFRvIII/JAK2/STAT3, and focal adhesion kinase (FAK) signaling pathways respectively. Interestingly, afatinib specifically inhibited EGFRvIII-cMET crosstalk in CSCs, resulting in decreased expression of Nanog and Oct3/4, and in combination with TMZ significantly decreased their self-renewal property in vitro. More interestingly, afatinib and TMZ combination significantly decreased the xenograft growth and progression compared to single drug alone. CONCLUSION: Our study demonstrated significant inhibition of GBM tumorigenicity, CSC maintenance in vitro, and delayed tumor growth and progression in vivo by combination of afatinib and TMZ. Our results warrant evaluation of this drug combination in EGFR and EGFRvIII amplified GBM patients.